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Scanning Electron Microscopy (SEM): An Introduction to the use of SEM for

character- ising the Surface Topology and Composition of Matter with Further
Applications

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Experimental Techniques In Condensed Matter Physics Instructor: Dr Marta Castro Lopez

Scanning Electron Microscopy (SEM): An Introduction to the use of SEM for character-
ising the Surface Topology and Composition of Matter with Further Applications

James A. Seyforth
Department of Natural & Mathematical Sciences, King’s College London, December 7th 2015

This investigation is both a brief introduction to the Scanning Electron Microscope, as well as an elaboration of
some fascinating potential applications. Three samples, including Nano-rods manufactured at King’s College Lon-
don, a beetle and pods of pollen were probed via SEM. High resolution images were produced for all three, show-
ing remarkable differentiations of surface topology at micro-nanoscopic scale. Also, the elemental composition of
the Nan-rods was found by using an INCA energy EDS Spectrometer from Oxford Instruments.

1-Introduction

The development the Scanning Electron Microscope

(SEM) started in 1926 when almost simultaneously,
Hans Busch discovered symmetric Electric and mag-
netic fields could act as particle lenses and de Broglie
developed his concept of corpuscle waves [1], where
he found the wavelength of an electron is given by
equation (1) (with added relativistic corrections) [2]:
ℎ ℎ ℎ 1
𝜆= = = (1)
𝑝 𝑚0 𝑣 √2𝑚0 𝑒𝑈 √1+ 𝑒𝑈
2𝑚0 𝑐2

Here 𝜆 is the electron wavelength (m), h is planck’s

constant (~6.6 x 10-34 J s), 𝑚0 is the rest mass of an
electron (~9.1 x 10 -31 kg), e is the electron charge
(~1.6 × 10-19C), U is the electric potential of the
source (V) and c is speed of light (~3.0 × 108 ms-1).
SEM operates by scanning a beam of these ‘wave-
like’ electrons, which have been focused to a fine
spot (as small as 1nm in diameter) [3] across the sur-
face of any sample prepared for probing. To Form an
image the interaction between the beam and the sam-
ple is monitored via a detector, which measures the
intensities of different areas of interaction, where the
most commonly used is the secondary electron sig-
nal.
Firstly, to create a focused beam of electrons, the
SEM utilises an electron optical column, where an
electron gun is fired through an array lenses that fo-
cus the beam, this is shown below in Figure 1. The
range of electric potentials used in this investigation
was 5-25 kV, which using (2) below gives electron ve-
locities of approximately 4.2 × 107 𝑚𝑠 −1 and 9.4 ×
107 𝑚𝑠 −1respectively. Equation (2) gives the electron
velocity as:
Figure 1: The SEM electron optical column can be seen be-
tween the electrons source and lens 3. The specimen
chamber sits inside the vacuum where the detector is also
housed by the collector system.
Optical column contained air at normal atmospheric
pressure, the electrons would immediately collide
with gas particles. Since this would render the SEM
completely ineffective, various degrees of vacuum
must be maintained throughout the entire device to
ensure the electrons can propagate to the sample, and
back to the detector [4]. Typical pressures are around
10-8 Pa near the electron source and sample.
The ability of SEM to achieve sub-nm resolu-
tion, which is far greater than any optical resolution,
is determined by the electron wavelength in (1),
which in this case, for 5-25kV gives 17 – 7.4 pm.
Equation (3) below defines the resolution of a system
as:
𝜆 𝜆
2𝑑 = = (3)
𝑛𝑠𝑖𝑛𝜃 𝑁𝐴

2𝑒𝑈 Where d is the size of the smallest feature the system

𝑣= √
𝑚 0
Where v is the velocity of the electron (ms-1) and all
the other parameters are the same as listed for (1).
Also, it is because the velocities calculated above are
respectively 14% and 31% of the speed of light that
we must add the relativistic corrections in equation
(1). The electrons travel extremely fast and if the
(2)
can resolve (m), n is the refractive index of the me-
dium, NA is the numerical aperture, 𝜃 is the half an-
gle subtended by the objective lens and 𝜆 is wave-
length of incident light(m) [5][6]. Using this equation
we see that for 5-25kV where 𝜆 = 17 – 7.4 pm, and
NA = 1, we get d = 8.5 – 3.7 pm. This shows the
smallest resolvable features are less sub-nm.
Experimental Techniques In Condensed Matter Physics
Finally, the detector must take into account the com-
plex electron-sample interaction volume that occurs
when the electrons reach the sample. There are two
types of interaction: Elastic electron scattering, where
incident electrons transfer no energy to the sample
and inelastic electron scattering, where electrons do
transfer energy to the sample. Below in figure 2 the
interaction volume of the incident beam can be seen:
Figure 2: Scattering circled in purple is elastic: no energy
transfer. Scattering circled in orange is Inelastic: energy
transfer. The Secondary electrons penetrate the least,
whilst the x-ray producing electrons penetrate deepest. [7][8]
To get an idea of the outcome of this interaction,
Monte Carlo [9] simulations can be run to determine
volume via using parameters such as sample thick-
ness and atomic number, as well as theoretical proba-
bility of each scattering event.
In this investigation, the scattering events of
most concern are the secondary electrons and the
characteristic x-ray emission. Secondary electrons
provide enhanced topographical resolution at low
electric potentials because the volume is small, mean-
ing the detector can more easily pin point the scatter-
ing locations [10]. X-rays are used for X-ray dispersive
spectroscopy because it is possible to characterise the
samples’ elements as each atom has a characteristic x-
ray emission energy associated to its unique energy
bands [11].
2 - Method and Collection of Data
In this investigation the first sample studied was a
collection of pollen grains and the pollen pods they
are contained in. The electron voltage was sat at
20kV, and the pollen pods had been prepared only
from above with gold in the z-direction, meaning that
it had incomplete coverage. Images were taken at 45⁰
and 90⁰ at various magnifications. Initial images at
x306 and x256 magnification can be seen below in
figure 3. The x252 is more blurred because it may of
been out of focus. The sample preparation is ex-
tremely important, as after bombardment with elec-
trons, non-conductive materials such as pollen will
Instructor: Dr Marta Castro Lopez
Figure 3: Left: Pollen grains in pods at x306 magnification.
Right: same pods at x252 magnification.
accumulate charge as a result of the build-up of elec-
trostatic fields near the surface. These fields will then
deflect more and more incident electrons as time
goes on resulting in less secondary electrons being
emitted [12]. By coating the sample in gold or plati-
num, and grounding them via an earth circuit, the
charge can be neutralised and the number of second-
ary electrons increased even further due to the high
value of these metals’ atomic number (Z). An in-
crease in Z results in an increase in positive protons
in the samples’ atoms, meaning that a greater force
will be felt by all incoming electrons and so the inter-
action volume will be smaller, and the number of sec-
ondary electrons greater [13][14].
The SEM used was a second hand machine
from Hitachi, originally built in the 1980s, yet it still
had a very high resolution of up to 2nm if the sample
was prepared correctly and the controls fine-tuned. It
is possible to scan the sample very slowly or very
quickly, but both have limitations: scanning fast
means the device is able to give the user immediate
visual feedback of the sample, but also means there’s
lower electron detection per unit time causing a lower
signal-to-noise ratio, resulting in grainy images. For a
slower scan rate, the user feedback is slow but the
electron detection per unit time is higher, meaning
there is a higher signal to noise ratio, increasing im-
age quality [15].
A beetle was also imaged at 20kV and had been
coated with gold. Images were taken of its eyes, an-
tenna and wing back plates. The bug was also tilted at
45⁰ and 90⁰. Two images of contrasting quality are
shown below in Figure 4, one of the antenna, the
other of a back plate:
Figure 4: Left: Beetle Antenna, shows grainy and
streaky hairs due to charging. Right: clear uniform
image of back plate structure aided by flat topology.
Experimental Techniques In Condensed Matter Physics Instructor: Dr Marta Castro Lopez

Clearly the antenna in Figure 4 has charging because

the streaks indicate that the detector is receiving a
non-uniform number of secondary electrons, causing
the intensity to vary greatly as seen in the image. Re-
ducing the electron energy via reduced voltage can
help, but in this case, the topology of the antennas is
extremely varied, with hairs and points; these points Figure 6: Slight streaks at the top of the image could indi-
cause further irregularities in electron detection, pre- cate charging due to non-uniform gold covering.
venting a high quality image [16]. of plants [18].
The Nano-rods were probed topographically as The Beetle’s eye, a fascination of many a biolo-
well as compositionally. This required magnification gist and nature enthusiasts, was also imaged at high
down to the nanometre scale rather than the micro- magnification and can be seen below in Figure 7:
metre. To achieve focus at this scale, a piece of dust
was located on the sample, and a rough focus was
achieved before magnifying. After this was approxi-
mately in focus, the magnification could be increased
and the focus again fine-tuned but now with respect
to the sample surface rather than the dust, achieving
a sharper image. Below in Figure 5 are two images of
the rods, one at 90⁰ and the other at 45⁰ with an in-
creased voltage of 110kV: Figure 7: The image shows the beetle’s eye with dirt
and cracks. The sample preparation may have caused
damage, or exposure to air over time may have re-
sulted in dirt particles adhering to the surface.
At x900 magnification, the beetle’s eye can be seen
clearly with sharpness and contrast. There appears to
be dirt from over exposure or bad sample prepara-
tion. However, this doesn’t stop one from speculat-
ing that this beetle appears to have circular ommatidi-
Figure 5: Left: Nano-rods at 90⁰ at x25’000 magnification.
Right: Nan-rods at 45⁰ with an x80’000 magnification at
ums (plural of ommatidia). An ommatidia is the lens
110 kV. mechanism that extends behind the surface of insects
with compound eyes. Below in Figure 8 the 3D struc-
Using equation (1), this gives an electron wavelength ture of the Beetles compound eye is conjectured [19]:
of ~3.5 pm of it can be seen that at 110kV, with the
smallest resolvable feature of ~1.8 pm using (2). This
gives around a 7 times greater resolution than at
25kV meaning the spacing between the Nano-rods
can be easily imaged at very high quality.
Using the INCA EDS Oxford instruments X-ray
dispersive spectrometer [17], a compositional measure-
ment was taken to determine the elements present in
the rods.

3 - Results and Analysis Figure 8: a collection ommatidiums have facets that

For the pollen, after the initial images in Figure 3
were measured, a higher magnification image was
produced with a slower scan rate, and a finer adjust-
ment of the brightness and contrast settings. The fi-
nal image can be seen below in Figure 6. The maze
like detail on the pollen appears much like the exine
ornamentation of many other pollen seeds. This pro-
tective wall defends the pollen grain against the envi-
ronment, as well as determines the way it attaches to
insect-pollinators and adheres to the stigmatic surface
are circular and are similar to those in Figure 7 for
the beetle’s eye, this forms a compound eye. [19]
Finally, the compositional measurement was taken
for the Nano-rods, the data acquired is shown below
in Figure 9. The graph clearly shows that Gold (Au)
emits from both the L and K electron shells (de-
scribed in Figure 10[20]) at high and low keV. Gold
was most likely used in the sample preparation and so
is not what the Nano-rods are actually made of.
Thus, The significant traces of Aluminium (Al),
Experimental Techniques In Condensed Matter Physics
Figure 9: Shows the relative amounts of elements that con-
stitute the Nano-rods by measuring the number of X-ray
photons detected per unit time for each X-ray energy.
Silicon (Si) and Calcium (Ca) suggest that these ele-
ments are what the rods are actually comprised of.
Figure 10: electrons drop form K, L or M shells producing
X-rays unique to each element and its structure.[20]
4 –Further Applications
The SEM can be used to image nano-scopic struc-
tures at high resolution. One fascinating application
is to the study of the ‘alien’ like bacterio phage virus.
They range from 24-200nm in size and have a unique
icosahedral head [21] containing all their DNA, they
also attack bacteria, intruding into the cells’ intracel-
lular domain, causing the bacteria’s replication system
to replicate more bacterio phages until bursting point
[22]. SEM has been used to characterise the structure
of this virus, two T2-bacterio phage viruses can be
seen below which were imaged using SEM:
Figure 11: T2-bacterio phage ; taken by ultra high resolu-
tion SEM, model UHS-t1 (Prof.K.Tanaka, Tottori Univer-
sity, Japan) (1988).
One interesting study discusses the potential uses of
phages for defence against bacterial infections, in
light of the continuing resistance to of bacteria to
anti-biotics. [23]
Instructor: Dr Marta Castro Lopez
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[1] A. Bogner, P.-H. Jouneau, G. Thollet, D. Basset,
C. Gauthier, A history of scanning electron micros-
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[2] An introduction to Electron Microscopy, FEI,
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[3] An introduction to Electron Microscopy, FEI,
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[4] An introduction to Electron Microscopy, FEI,
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[6] Marta Castro-Lopez, an introduction
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[9] Frank Krumeich, Properties of Electrons, their
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m=1596&cnt=3340
 Experimental Techniques In Condensed Matter Physics Instructor: Dr Marta Castro Lopez

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[17]https://www.brandonu.ca/micro-
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[18] http://www2.le.ac.uk/departments/genet-
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[21]http://www.nsf.gov/od/lpa/news/02/pr0207im
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[23] Sarah O'Flaherty, R. Paul Ross, Aidan Coffey,

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July 2009

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