Electron Microscope

1. SCANNING ELECTRON MICROSCOPY
AND TRANSMISSION ELECTRON
MICROSCOPY.

2. HISTORY
• The word microscope is derived from the Greek mikros (small)
and skopeo (look at).
• One of the earliest instruments for seeing very small objects was
made by the van Leeuwenhoek (1632-1723) and consisted of a
powerful convex lens and an adjustable holder for the object being
studied.

3. • With this remarkably simple microscope, Van Leeuwenhoek may
well have been able to magnify objects up to 400x; and with it he
discovered protozoa, spermatozoa, and bacteria, and was able to
classify red blood cells by shape.
• The limiting factor in Van Leeuwenhoek’s microscope was the
single convex lens. The problem can be solved by the addition of
another lens to magnify the image produced by the first lens and
this compound microscope – consisting of an objective lens and
an eyepiece together with a means of focusing, a mirror or a
source of light and a specimen table for holding and positioning
the specimen – is the basis of light microscopes today.

4. Resolving power of human eye
0.2 mm apart. If the points are closer together, they will
appear as a single point. This distance is called the resolving
power or resolution of the eye.
For example, try looking at a newspaper picture, or one in a
magazine, through a magnifying glass. You will see that the
image is actually made up of dots too small and too close
together to be separately resolved by your eye alone. The same
phenomenon will be observed on an LCD computer display or
flat screen TV when magnified to reveal the individual
“pixels” that make up the image

5. CLASSIFICATION OF MICROSCOPES
Microscopes can be classified as one of three basic types:
1) optical,
2) charged particle (electron and ion), or
3)scanning probe.
Optical microscopes are the ones most familiar to everyone from
the high school science lab or the doctor’s office. They use
visible light and transparent lenses to see objects as small as
about one micrometer (one millionth of a meter), such as a red
blood cell (7 μm) or a human hair (100 μm).

6. Electron and ion microscopes use a beam of charged particles instead
of light, and use electromagnetic or electrostatic lenses to focus the
particles. They can see features as small a tenth of a nanometer (one
ten billionth of a meter), such as individual atoms.
Scanning probe microscopes use a physical probe (a very small,
very sharp needle) which scan over the sample in contact or
near-contact with the surface. They map various forces and
interactions that occur between the probe and the sample to
create an image. These instruments too are capable of atomic
scale resolution.

7. Modern light microscope:
Has a magnification of about 1000x and enables the eye to resolve
objects separated by 200 nm.
Scientists realized that the resolving power of the microscope was not
only limited by the number and quality of the lenses, but also by the
wavelength of the light used for illumination. With visible light it was
impossible to resolve points in the object that were closer together than a
few hundred nanometers.
Other measures such as using light with shorter wavelengths (blue or
ultraviolet)or immersing specimens in medium having high refractive
index such as oil gave some improvement but only under 100 nm.

8. • In the 1920s, it was discovered that accelerated electrons behave in
vacuum much like light. They travel in straight lines and have wavelike
properties, with a wavelength that is about 100,000 times shorter than that
of visible light.
• Furthermore, it was found that electric and magnetic fields could be used
to shape the paths followed by electrons similar to the way glass lenses
are used to bend and focus visible light.
• Ernst Ruska at the University of Berlin combined these characteristics
and built the first transmission electron microscope (TEM) in 1931.
• For this and subsequent work on the subject, he was awarded the Nobel
Prize for Physics in 1986.
• The first electron microscope used two magnetic lenses, and three years
later he added a third lens and demonstrated a resolution of 100 nm, twice
as good as that of the light microscope.
• Today, electron microscopes have reached resolutions of better than 0.05
nm, more than 4000 times better than a typical light microscope and
4,000,000 times better than the unaided eye.

10. Scanning electron microscopy
• It is not completely clear who first proposed the principle of
scanning the surface of a specimen with a finely focused electron
beam to produce an image.
• The first published description appeared in 1935 in a paper by the
German physicist Max Knoll.
• Although another German physicist, Manfred von Ardenne,
performed some experiments with what could be called a scanning
electron microscope (SEM) in 1937.
• It was not until 1942 that three Americans, Zworykin, Hillier, and
Snijder, first described a true SEM with a resolving power of 50 nm.
Modern SEMs can have resolving power better than 1 nm

11. Principle of SEM:
• The specimen is bombarded by a convergent electron beam, which is
scanned across the surface.
• This electron beam generates a number of different types of signals,
which are emitted from the area of the specimen where the electron
beam is impinging The induced signals are detected and the intensity
of one of the signals (at a time) is amplified and used to as the intensity
of a pixel on the image on the computer screen.
• The electron beam then moves to next position on the sample and the
detected intensity gives the intensity in the second pixel and so on.

12. Scanning electron microscope

13. • In the first part of this laboratory session the image formation using
two types of signals, secondary electrons (SE) and backscatter electron
(BE) will be studied.
• The SEM can be operated in many different modes where each mode
is based on a specific type or signal. The choice of operating mode
depends on the properties of the sample and on what features one
wants to investigate. The modes are as follows:
a) Secondary electrons (SE)
b) Backscattered electrons (BE)
c) Electron backscattered diffraction (EBSD)
d) X-ray
e) Absorbed current
f) Transmitted electrons
g) Beam induced conductivity
h) Cathodoluminescence
Mainly
emphasized

14. a) Secondary electron mode:-
– Electrons with energies between 0 – 30 eV are detected and used to
form the image. These electrons are knocked out from the specimen
by the incident electron beam and come from a layer within 5 nm of
the surface.
b) Backscattered electron mode:-
– Electrons with energies with energies ranging from a few keV to the
energy of the incident electrons (typically 15 – 30 keV) are detected.
– Such electrons are electrons from the electron beam that are
elastically scattered back from the sample.
– They scattering takes place in a volume extending down to 0.5μm
below the surface and therefore gives information also about the
“bulk” properties of the material

15. c) Electron backscattered diffraction mode:-
It is an easy and rapid technique to study crystallographic orientation,
microtexture, phase distribution and grain characterization. The method is
based on the analysis of diffraction patterns from flat bulk samples.
– One main advantages of the EBSD technique is that the EBSD detector
is inserted in the SEM allowing recording of both images and patterns
from the same region
– Another advantage is that the specimen-detector distance can be made
relatively short, so it is possible to record diffraction pattern covering a
relatively large range of diffraction angles and therefore make the
analysis more accurate.

16. Optical microscope vs. SEM

18. Principle of TEM:
• The transmission electron microscope can be compared with a slide
projector.
• In a slide projector light from a light source is made into a parallel beam
by the condenser lens; this passes through the slide (object) and is then
focused as an enlarged image onto the screen by the objective lens.
• In the electron microscope, the light source is replaced by an electron
source, the glass lenses are replaced by magnetic lenses, and the
projection screen is replaced by a fluorescent screen, which emits light
when struck by electrons, or, more frequently in modern instruments, an
electronic imaging device such as a CCD (charge-coupled device)
camera.

19. The whole trajectory from source to screen is under vacuum
and the specimen (object) has to be very thin to allow the
electrons to travel through it. The sample must be pre-treated with
heavy metals which by preference bind ("stain") to certain
characteristic structures, like membranes, proteins and DNA.
Not all specimens can be made thin enough for the TEM.
Alternatively, if we want to look at the surface of the specimen,
rather than a projection through it, we use a scanning electron or
ion microscope.

20. • Once in the TEM the object is bombarded by a beam of electrons,
the so-called primary electrons.
• In areas in the object where these electrons encounter atoms with a
large (heavy) atomic nucleus (e.g. the nuclei of the heavy metals of
the pretreatment), they rebound. Electrons are also repulsed (or
absorbed) in areas where the material is relatively condense or
thick.
• However, in regions where the material consists of lighter atoms or
where the specimen is thinner or less concentrated, the electron are
able to pass through. Eventually the traversing electrons
(transmission) reach the scintillator plate at the base of the column
of the microscope.

21. • The scintillator contains material (e.g. phosphor compounds) that can
absorb the energy of the stricking incoming electrons and convert it to
light flashes. The contrasted image that is formed on this plate
corresponds with the selective pattern of reflection or permission of
electrons, depending on the local properties of the object.
• Thus, one can see for example where cytoskeletal elements and
membranes are located because the corresponding area remains dark,
whereas the cytosol around these structures appears as light. In practice
the bombarding electrons are focused to a bundle onto the object.
• The fine pattern of exiting electrons leaving the object is then greatly
enlarged by electromagnetic lenses: a many times enlarged projection
image is the result.

22. The transmission electron microscope is made up of:
 The illuminating system consists of the electron gun and condenser lenses
that give rise to and control the amount of radiation striking the specimen.
 A specimen manipulation system composed of the specimen stage,
specimen holders, and related hardware is necessary for orienting the thin
specimen outside and inside the microscope.
 The imaging system includes the objective, intermediate, and projector
lenses that are involved in forming, focusing, and magnifying the image on
the viewing screen as well as the camera that is used to record the image.
 A vacuum system is necessary to remove interfering air molecules from the
column of the electron microscope. In the descriptions that follow, the
systems will be considered from the top of the microscope to the bottom.

23. Major Column Components of the TEM*
Component Synonyms Function of Components
Illumination System
Electron Gun Gun, Source Generates electrons and provides first
coherent crossover of electron beam
Condenser Lens 1 C1, Spot Size Determines smallest illumination spot
size on specimen
Condenser Lens 2 C2, Brightness Varies amount of illumination on
specimen—in combination with C1
Condenser Aperture C2 Aperture Reduces spherical aberration, helps
control amount of illumination striking
specimen

24. Specimen Manipulation
System
Synonyms Function of Components
Specimen Exchanger Specimen Air Lock Chamber and mechanism for
inserting specimen holder
Specimen Stage Stage Mechanism for moving specimen
inside column of microscope
Imaging System
Objective Lens — Forms, magnifies, and focuses first
image
Objective Aperture — Controls contrast and spherical
aberration
Intermediate Lens Diffraction Lens Normally used to help magnify
image from objective lens and to
focus diffraction pattern
Intermediate Aperture Diffraction Aperture, Field
Limiting Aperture
Selects area to be diffracted
Projector Lens 1 P1 Helps magnify image, possibly used
in some diffraction work
Projector Lens 2 P2 Same as P1

25. Observation and
Camera Systems
Synonyms Function of Components
Viewing Chamber — Contains viewing screen for
final image
Binocular Microscope Focusing Scope Magnifies image on viewing
screen for accurate focusing
Camera — Contains film for recording

26. Differance between TEM and SEM
TEM SEM
It uses a high-powered beam to essentially
shoot electrons through the object.
It doesn’t use a concentrated electron
beam to penetrate the object
The beam goes through the object. Some of
the electrons pass all the way through;
others hit molecules in the object and
scatter. The modified beam then passes
through an objective lens, a projector lens
and onto a fluorescent screen where the
final image is observed, the pattern of
scatter gives the observed a
comprehensive view of the interior of the
object.
It scans a beam across the object.
During the scanning the beam loses
energy in different amounts according
to the surface it is on. A scanning
electron microscope measures the lost
energy to create a three-dimensional
picture of the surface of an object.

27. A Transmission Electron
Microscope (TEM) produces a 2D
image of a thin sample, and has a
maximum resolution of ×500000.
A Scanning Electron Microscope
(SEM) produces a 3D image of a
sample by 'bouncing' electons off and
dectecting them at multiple detectors.
It has a maximum magnification of
about ×100000.
Image of pollen