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Microscopy
Unresolved
Partially resolved
Resolved
Optical microscopy - limitations
OM can only image dark or strongly
refracting objects effectively
.
Out of focus light from points outside the
focal plane reduces image clarity.
Compound optical microscopes are limited
in their ability to resolve fine details by the
properties of light and the refractive
materials used to manufacture lenses. A
lens magnifies by bending light..
Optical microscopes are restricted in their ability to
resolve features by a phenomenon called diffraction
which, based on the numerical aperture AN of the
optical system and the wavelengths of light used (λ),
sets a definite limit (d) to the optical resolution.
Assuming that optical aberrations are negligible, the
resolution (d) is given by:
Scanning
Electron
Microscope
Remember that there are 1000 micrometers (µm) in 1 mm and
1000 nanometers (nm) in 1 µm.
17
Light Microscopes
Sources of Illumination
• Light microscopes use a beam of light for
illumination and include fluorescence and
confocal microscopes
Electron Microscopy
• beams of electrons
are used to
produce images
• wavelength of
electron beam is
much shorter than
light, resulting in
much higher
resolution
29
Types of Electron Microscope
• Transmission Electron Microscope (TEM) uses a
wide beam of electrons passing through a thin
sliced specimen to form an image. This
microscope is analogous to a standard upright or
inverted light microscope
32
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EM
33
Transmission Electron Microscopy (TEM)
Ultrathin sections of
specimens
Light passes through
specimen, then an
electromagnetic lens,
to a screen or film
Specimens may be
stained with heavy
metal salts
Specimen Preparation
• analogous to procedures used for
light microscopy
• for transmission electron
microscopy, specimens must be cut
very thin
• specimens are chemically fixed and
stained with electron dense material
36
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
37
Electron Microscopy - definition and types
• developed in the 1930s that use electron beams instead of light.
• because of the much lower wavelength of the electron beam
than of light, resolution is far higher.
TYPES
• Transmission electron microscopy (TEM) is principally
quite similar to the compound light microscope, by sending an
electron beam through a very thin slice of the specimen. The
resolution limit (in 2005) is around 0.05 nanometer.
Projector Specimen
Eyepiece CRT
Cathode Ray
Tube
detector
OM TEM SEM
Transmission Electron
Microscope
Illumination source is
beam of electrons from
tungsten wire
Electromagnetic lenses
perform same function
as glass lenses in LM
Higher resolution and
higher magnification of
thin specimens
FEI Tecnai 20
For TEM, since the electrons
need to penetrate the specimen, it
must be very thin (< 100 nm)
Comparison of TEM and LM
© OCR 2016
•All rays from a point in the object are gathered by the lens and
converge to a point in the image.
•All parallel rays are focused in the focal plane.
•The back focal plane of the objective lens contains groupings of
rays that have left the object at the same angle.
•The back focal plane contains the diffraction pattern of the sample.
•Diffraction pattern and image are both formed in the imaging
process
•The intermediate lens is then focused on either the image plane
(for the image), or the back focal plane (for the diffraction pattern).
Dark Field – Advantages and Disadvantages
•Advantages and disadvantages of Dark Field Imaging :-
•Advantages
•Provides high contrast for examining molecules with
very low contrast such as DNA.
•For crystalline objects, specific diffraction spots can be
selected in the back focal plane of the objective lens in
order to form a dark field image only from the electrons
scattered by a chosen set of crystal planes.
•Disadvantages
•More difficult to focus and correct for astigmatism since
phase contrast is not present.
•Image brightness is low, since the objective aperture
transmits only a small fraction of the scattered beam,,
•Longer exposure times needed to get good
photographic images.
•Consequently, specimens are subjected to greater
levels of radiation damage.
Imaging Modes
Bright field/dark field depends on the aperture position. Modern way for
diffraction tilts the beam instead of moving the aperture
Magnification in TEM
(1-50KeV)
Polymer
Polymercrystalline
crystalline
structure
structureand
and
morphology
morphology
Application
Polymer
Polymercomposition
composition Distribution
Distributionof
ofdispersed
dispersed
phase
phase
Examples
Graphene
Carbon
nanotube
PE/SWNT NHSK
Transmission Electron Microscopy
(TEM)
Neuron growing on astroglia Black Ant
House Fly
House Fly
Human stem
cells
Neurons
CNS
Transmission Electron Microscopy
(TEM)
View inside cell via sections
magnification 120,000 X
50,000X
Conventional TEM Micrographs
Bacteria in cell
Apoptosis
Skin
To produce the SEM image, the electron beam is swept across the area being
inspected, producing many such signals. These signals are then amplified, analyzed,
and translated into images of the topography being inspected. Finally, the image is
shown on a CRT.
Scanning Electron Microscopy (SEM)
Resolution
• depends on the size of the electron spot, which in turn
depends on the magnetic electron-optical system which
produces the scanning beam.
• is not high enough to image individual atoms, as is
possible in the TEM … so that, it is 1-20 nm
Advantages of Using SEM over OM
The SEM has a large depth of field, which allows a large amount of the
sample to be in focus at one time and produces an image that is a good
representation of the three-dimensional sample. The SEM also produces
images of high resolution, which means that closely features can be
examined at a high magnification.
Magnification?
Resolution?
Image Formation in SEM
M= C/x
-
A 10cm
e
beam
Detector
10cm
Amplifier
92
Scanning Electron Microscopy (SEM)
• An electron gun produces a beam of electrons that
scans the surface of a whole specimen.
• Secondary electrons emitted from the specimen
produce the image.
Figure 3.9b
Electron beam
produced here
Beam passes down the
microscope column
Cross section of
electromagnetic Electron beam now tends to
lenses diverge
But is converged by
electromagnetic lenses
Sample
96
SEM components
What is SEM
Column
TV Screens
Sample
Chamber
The SEM is designed
for direct studying of
the surfaces of solid
objects
Cost: $0.8-2.4M
A more e- beam
detailed
look α
inside
Source: L. Reimer,
“Scanning Electron
Microscope”, 2nd Ed.,
Springer-Verlag, 1998, p.2
Cathode Ray Tube (CRT) accelerates electrons towards the
phosphor coated screen where they produce flashes of light
upon hitting the phosphor. Deflection coils create a scan
pattern forming an image in a point by point manner
Color CRT?
Image Magnification
W or LaB6 Filament
Thermionic or Field Emission Gun
Thermionic Emission Gun
• A tungsten filament
heated by DC to
approximately 2700K or
LaB6 rod heated to around
2000K
• A vacuum of 10-3 Pa (10-4 -
Pa for LaB6) is needed to
prevent oxidation of the
filament +
• Electrons “boil off” from
the tip of the filament
• Electrons are accelerated
by an acceleration voltage
of 1-50kV
Source of Electrons
Thermionic Gun E: >10MV/cm
T: ~1500oC
W
Filament
(5-50µm)
(5nm)
W hairpin FEG
LaB6 crystal
Thermionic Sources
Increasing the filament current will increase the beam current but
only to the point of saturation at which point an increase in the
filament current will only shorten the life of the emitter
Beam spot image at different stage of heating
Magnetic Lenses
• Condenser lens – focusing
determines the beam current
which impinges on the sample.
• Objective lens – final probe
forming
determines the final spot size of
the electron beam, i.e., the
resolution of a SEM.
Electromagnetic Lenses
Recall the right hand rule electron will move in a helical path
spiralling towards the centre of the magnetic field
Electromagnetic lens
Why Need a Vacuum?
When a SEM is used, the electron-optical column and
sample chamber must always be at a vacuum.
2. Other gas molecules, which could come from the sample or the
microscope itself, could form compounds and condense on the
sample. This would lower the contrast and obscure detail in the
image.
The Condenser Lens
• For a thermionic gun, the diameter of
the first cross-over point ~20-50µm
• If we want to focus the beam to a size
< 10 nm on the specimen surface, the
magnification should be ~1/5000, which
is not easily attained with one lens (say,
the objective lens) only.
• Therefore, condenser lenses are added
to demagnify the cross-over points.
The objective lens
The objective lens aperture
F = -e(v x B) p
q
Lens formula: 1/f = 1/p + 1/q
Demagnification: M = q/p
f ∝ Bo 2
Demagnification:
M = f/L
Condenser-lens system
Objective
lens
Sample stage
Topographical Contrast
Everhart-Thornley
SE Detector
e-
SE Scintillator
light pipe PMT
Dark
sample Quartz
Faraday window
cage +200V +10kV
Photomultiplier
tube
+ +
The most common design is a four quadrant solid state detector that is positioned
directly above the specimen
Example of an image using a scanning electron microscope and
secondary electrons
Grain containing
of silica so it is
darker
Grain containing
Here the differing contrast of the
titanium so it is
grains tells us about composition
whiter
So how does this work – telling composition from
backscattered electrons?
This is a characteristic
packet of energy and can
tell us what element we
Inelastic scattering are dealing with
Backscattered Electrons (BSE)
Primary
Pear shape
5µm
a b
x Low M High M
Large x small x
40µm 7µm
1.2µm 15000x
2500x
The magnification is simply the ratio of the length of the scan C on the
Cathode Ray Tube (CRT) to the length of the scan x on the specimen. For a
CRT screen that is 10 cm square:
M= C/x = 10cm/x
Increasing M is achieved by decreasing x.
M x M x
100 1 mm 10000 10 µm
1000 100 µm 100000 1 µm
Resolution Limitations
Ultimate resolution obtainable in an SEM image can be
limited by:
P=D/Mag = 100um/Mag
Mag P(µm) Mag P(nm)
P-pixel diameter on specimen surface 10x 10 10kx 10
D-pixel diameter on CRT, Mag-magnification 1kx 0.1 100kx 1
Resolution of Images: II
• The optimum condition for imaging is when
the escape volume of the signal concerned
equals to the pixel size.
Resolution of Images: III
• Signal will be weak if escape volume,
which depends on beam size, is smaller
than pixel size, but the resolution is still
achieved. (Image is ‘noisy’)
Resolution of Images: IV
• Signal from different pixel will overlap
if escape volume is larger than the
pixel size. The image will appeared
out of focus (Resolution decreased)
Resolution of Images: V
In extremely good SEM, resolution can be a few nm. The
limit is set by the electron probe size, which in turn depends
on the quality of the objective lens and electron gun.
Pixel diameter on Specimen
Magnification µm nm
10 10 10000
100 1 1000
1000 0.1 100
10000 0.01 10
100000 0.001 1
Depth of Field
Depth of Field
4x105W
D= (µm)
AM
To increase D
Decrease aperture size, A
Decrease magnification, M
Increase working distance, W (mm)
SE Images
Image Contrast
Image contrast, C
is defined by
SA-SB ∆S
C= ________ = ____
SA SA
162
The Scanning Electron Microscope is analogous to the
stereo binocular light microscope because it looks at
surfaces rather than through the specimen.
Main Applications
• Topography
The surface features of an object and its texture
(hardness, reflectivity… etc.)
• Morphology
The shape and size of the particles making up the
object (strength, defects in IC and chips...etc.)
• Composition
The elements and compounds that the object is
composed of and the relative amounts of them
(melting point, reactivity, hardness...etc.)
• Crystallographic Information
How the grains are arranged in the object
(conductivity, electrical properties, strength...etc.)
SE Images - Topographic Contrast
1µm
2µm