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 Microscopy Methods in
Nanomaterials Characterization

Volume 1

Edited by
Sabu Thomas
Raju Thomas
Ajesh K. Zachariah

Raghvendra Kumar Mishra

Elsevier
Radarweg 29, PO Box 211, 1000 AE Amsterdam, Netherlands
The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom
50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States
Copyright © 2017 Elsevier Inc. All rights reserved.
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Publisher (other than as may be noted herein).

Notices
Knowledge and best practice in this field are constantly changing. As new research and experience
broaden our understanding, changes in research methods, professional practices, or medical treatment
may become necessary.
Practitioners and researchers may always rely on their own experience and knowledge in evaluating
and using any information, methods, compounds, or experiments described herein. In using such
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parties for whom they have a professional responsibility.
To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume
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negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas
contained in the material herein.

Library of Congress Cataloging-in-Publication Data

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ISBN: 978-0-323-46141-2

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List of Contributors
Rameshwar Adhikari
Tribhuvan University, Kathmandu, Nepal
Gudimalla AppaRao
Acharya Nagarjuna University, Guntur, India
Zahava Barkay
Tel-Aviv University, Tel-Aviv, Israel
Anjali Bishnoi
Shroff S. R. Rotary Institute of Chemical Technology, Gujarat, India; Indian Institute of
Technology, New Delhi, India
Mauro Ciappa
ETH Zurich, Zurich, Switzerland
Darshan B. Desai
Texas Tech University, Lubbock, TX, United States
Yue Fang
Zhejiang University, Hangzhou, People’s Republic of China
Gurram Giridhar
Acharya Nagarjuna University, Guntur, India
Sven Henning
Fraunhofer IMWS, Halle (Saale), Germany
Zhong Hu
South Dakota State University, Brookings, SD, United States
Yujia Huang
Zhejiang University, Hangzhou, People’s Republic of China
Arkady M. Ilyin
Al-Farabi Kazakh National University, Almaty, Republic of Kazakhstan
Nirav Joshi
University of São Paulo, São Paulo, Brazil
Cuifang Kuang
Zhejiang University, Hangzhou, People’s Republic of China
Sunil Kumar
Shroff S. R. Rotary Institute of Chemical Technology, Gujarat, India; Indian Institute of
Technology, New Delhi, India
Shaocong Liu
Zhejiang University, Hangzhou, People’s Republic of China
Xu Liu
Zhejiang University, Hangzhou, People’s Republic of China

xiii
xiv List of Contributors

R.K.N.R. Manepalli
Hindu College, Machilipatnam, India
Raghvendra Kumar Mishra
Mahatma Gandhi University, Kottayam, Kerala, India
Aristeidis Papagiannopoulos
National Hellenic Research Foundation, Athens, Greece
Sabu Thomas
Mahatma Gandhi University, Kottayam, Kerala, India
Thomas Walther
University of Sheffield, Sheffield, England, United Kingdom
Ajesh K. Zachariah
Mar Thoma College, Tiruvalla, Kerala, India
Editor Biographies
Professor (Dr.) Sabu Thomas is the Director of International and
Interuniversity Center for Nanoscience and Nanotechnology, Mahatma
Gandhi University, Kottayam, Kerala, India. He is also a full professor of
Polymer Science and Engineering and School of Chemical Science of the
same University. He is a fellow of many professional bodies. Professor
Thomas has co-authored many papers in international peer-reviewed
journals in the area of polymer processing. He has organized several
international conferences. Professor Thomas’s research group is in
specialized areas of polymers, which includes polymer blends, fiber-filled
polymer composites, particulate-filled polymer composites and their
morphological characterization, aging, and degradation, pervaporation
phenomena, sorption, and diffusion, interpenetrating polymer systems,
recyclability and reuse of waste plastics and rubbers, elastomeric cross-
linking, and dual porous nanocomposite scaffolds for tissue engineering.
Professor Thomas’s research group has extensive exchange programs with
different industries, research, and academic institutions all over the world
and is performing world-class collaborative research in various fields. The
Professor’s Center is equipped with various sophisticated instruments and
has established state-of-the-art experimental facilities which cater to the
needs of researchers within the country and abroad. He has more than 700
publications, 50 books, H Index-78 and 3 patents to his credit. He is a
reviewer to many international journals. Professor Thomas has attained
5th Position in the list of Most Productive Researchers in India in 2008e16.

Professor (Dr.) Raju Thomas is currently Vice Chancellor of Middle East

University FZE, Al Hamra, Ras Al Khaimah, United Arab Emirates.
Dr. Thomas started his Professorship from the Research and Postgraduate
Department of Chemistry, Mar Thoma College, Thiruvalla-3, Kerala,
India. Dr. Thomas procured his PhD under the supervision of Professor
(Dr.) Sabu Thomas, Director of International and Interuniversity Center
for Nanoscience and Nanotechnology, Mahatma Gandhi University,
Kottayam, Kerala, India. He has extensive research experience in
Nanoscience and Nanotechnology. He has 12 years of research experience
in the Organic Chemistry and Polymer Chemistry laboratories of the
School of Chemical Sciences, Mahatma Gandhi University, Kottayam,
Kerala, India. He has also worked in the laboratory of Applied Rheology
and Polymer processing of Katholieke University, Leuven, Belgium, and
in the laboratory at Leibniz Institute of Polymer Research, Dresden,

xv
xvi Editor Biographies

Germany. He has widely studied the kinetics of curing, morphology

development, and structural characteristics of in situ-cured
nanocomposites based on epoxy resin and reactive rubbers. His research is
reflected in his six published research articles in international journals, and
additional articles which are currently under review. In addition, many
articles have been published in popular journals. He has co-authored many
chapters and is co-editor of a book entitled Micro and Nanostructured
Epoxy/Rubber Blends which was recently published by Wiley and Sons.
He has attended many national and international seminars/conferences and
presented many research papers. He is an approved research guide in
Chemistry at Mahatma Gandhi University, Kottayam, India and has
availed projects from University Grants Commission (UGC), Department
of Science and TechnologyeScience and Engineering Research Board
(DSTeSERB) and Kerala Science Council for Science, Technology, and
Environment (KSCSTE).

Dr. Ajesh K. Zachariah is working as Assistant Professor in the

Department of Chemistry, Mar Thoma College, Kerala, India. He has
many publications in the field of materials chemistry, and polymer
nanocomposites and has one national patent. He is an expert in
sophisticated techniques such as Atomic Force Microscopy (AFM), X-ray
diffraction Technique (XRD), Gas Permeability Tester, and Dynamic
Mechanical Analyzer (DMA). He has many years’ experience in the field
of nanoscience and nanotechnology.

Raghvendra Kumar Mishra is currently working as Senior Research

Fellow at the International and Interuniversity Center for Nanoscience and
Nanotechnology, Mahatma Gandhi University, India. He has received
India’s most prestigious Visvesvaraya Research Fellowship, and he is
currently serving as Visvesvaraya Fellow. He has widely studied the
processing of blends, in situ generation micro- and nanofibrillar
composites, electromagnetic shielding effect of nanocomposites,
decorating and alignment of carbon nanotubes, and thermal, dynamic
mechanical, and structural relationships in polymer blends and
nanocomposites. He has won several awards from different organizations
and technology events. He is serving as reviewer in many international
journals, for example, Environmental Chemistry Letters (Springer). He has
research experience in Mechanical Engineering, Materials Science and
Technology, and Nanoscience and Nanotechnology. His areas of research
are multidisciplinary, which include thermodynamics, heat transfer,
refrigeration and air-conditioning, fluid mechanics, machine design, solid
mechanics, machine theory, power plant engineering, metal and ceramic
 Editor Biographies xvii

processing. In addition, he specializes in polymers, which include polymer

recycling, polymer blends, fiber-filled polymer composites, particulate-
filled polymer composites and their morphological characterization, aging
and degradation, nanomaterials e.g., metallic, metallic oxide, carbon
nanotubes, graphene, conducting polymer blends, composites and
nanocomposites, biodegradable polymer blends and composites. He has
expertise in sophisticated characterization techniques such as dynamic
mechanical analyzer, differential scanning calorimetry, thermogravimetric
analysis, spectroscopy, vector network analyzer, scanning electron
microcopy, and atomic force microscopy (AFM).
 Scanning Electron Microscopy, ESEM,
Chapter
1
and X-ray Microanalysis
Sven Henning1, Rameshwar Adhikari2
1
Fraunhofer IMWS, Halle (Saale), Germany; 2Tribhuvan University, Kathmandu, Nepal

CHAPTER OUTLINE
1.1 Introduction 2
1.2 Scanning Electron Microscopy 2
1.2.1 Principle 2
1.2.2 Vacuum System 3
1.2.3 Electron Beam Generation and Electron Optics 5
1.2.3.1 Electron Beam Generation 5
1.2.4 Signals, Detectors, and Image Formation 8
1.2.5 Sample Preparation 12
1.2.5.1 Mounting 12
1.2.5.2 Fracture Surfaces 13
1.2.5.3 Microtomed Sections and Cross Sections 13
1.2.5.4 Target Preparation 14
1.2.5.5 Focused Ion Beam 14
1.2.5.6 Drying 15
1.2.5.7 Conductive Coating 15
1.2.6 Examples 16
1.3 Energy Dispersive X-ray Microanalysis 18
1.3.1 Signal Generation and Detection 18
1.3.2 Examples 19
1.4 Environmental Scanning Electron Microscopy 20
1.4.1 Principle and Advantages 21
1.4.2 Instrumentation 22
1.4.3 Examples 22
1.5 Cryo Scanning Electron Microscopy 25
1.5.1 Instrumentation 25
1.5.2 Examples 25

Microscopy Methods in Nanomaterials Characterization. http://dx.doi.org/10.1016/B978-0-323-46141-2.00001-8

Copyright © 2017 Elsevier Inc. All rights reserved. 1
2 CHAPTER 1 Scanning Electron Microscopy, ESEM, and X-ray Microanalysis

1.6 Low-Voltage and High-Resolution Scanning Electron Microscopy 26

1.6.1 Low-Voltage Scanning Electron Microscopy and Inlens Detectors 26
1.6.2 Helium Ion Microscopy 28
References 28

1.1 INTRODUCTION
Scanning electron microscopy (SEM) is probably one of the most versatile
tools for the analysis of micro- and nanostructures with a wide range of
applications. SEM techniques provide information on the topography and
composition of surfaces by the collection and processing of signals that
are generated by a sharp electron probe within a certain interaction volume.
The main advantages can be summarized as follows: First, SEM provides
the imposing visualization of structures (“image to imagination”). This is
due to the capability to produce pictures with high plasticity and almost ste-
reoscopic appearance, and, depending on imaging conditions, a large depth
of focus even for samples with bold relief. Second, there is the huge range
of adjustable magnifications from reading-glass magnification (approxi-
mately 10 times), e.g., for overview mapping of large sample areas or to
identify regions of interest, and the possibility to zoom into the range of
some nanometers, depending on the instrument (standard vs. high resolu-
tion) and sample properties. Third, there is the analytical power to achieve
information on the material composition by the selection of signals origi-
nating from specific interactions, as there are, for instance, characteristic
X-ray quanta or backscattered electrons.
The chapter is based on a lecture series addressed to students as well as to
SEM users from academia and industry. The aim of the lecture is to demon-
strate the opportunities and limitations of SEM applications. It should
enable the potential user (or customer of SEM service facilities) to formu-
late the aim of the investigation more precisely, identify proper sample
preparation techniques, and perform a qualified image interpretation based
on the knowledge of general principles of the image formation and basic
mechanisms of contrast generation.

1.2 SCANNING ELECTRON MICROSCOPY

1.2.1 Principle
Comparable to reflected-light optical microscopy, SEM is a method to image
surfaces of a sample. In the years 1937e38, the German physicist and inventor
 1.2 Scanning Electron Microscopy 3

Manfred von Ardenne constructed an instrument that allowed the generation of

magnified images of a sample by scanning the surface using an electron beam
(the electron probe) in a line-by-line manner. The signals that are evoked by
the interaction of the electron probe with a certain interaction volume close to
the sample surface can be amplified and are translated to bright spots of lower
or higher intensity, depending on the signal intensity, in a simultaneous line-
by-line manner on a cathode ray tube (CRT) (Fig. 1.1). The magnification of
the so-called “Raster-Elektronenmikroskop” is defined by the ratio of the
frame size of the scanned sample area and the size of the CRT monitor. The
development of the instrument was prepared, on one hand, by the invention
of the transmission electron microscope by Knoll and Ruska a few years earlier
(1931, Nobel Prize 1986). On the other hand, von Ardenne was already known
for his a pioneering in electronic television, presenting the first television sys-
tem using a CRT in 1931.
Today, SEM has evolved into a powerful, versatile tool used in many fields
of nanotechnology. The range of SEM magnification is shown in Fig. 1.2:
The maximum resolution that can be achieved is around 1 nm, depending on
the setup and quality of the instrument, operating parameters, and properties
of the sample material itself.

1.2.2 Vacuum System

Because SEM works with electrons that have to be generated, accelerated,
focused, and guided toward the sample, the whole SEM column is set under

n FIGURE 1.1 Historic photograph of Manfred von Ardenne’s first “Raster-Elektronenmikroskop hoher Auflösung” [1] and schematic drawing of the SEM function [2].
4 CHAPTER 1 Scanning Electron Microscopy, ESEM, and X-ray Microanalysis

10-11

10-10

10-9

10-8

10-7

10-6

10-5

10-4

10-3
m

resolution
10 pm

100 pm

1 nm

10 nm

100 nm

1 µm

10 µm

100 µm

1 mm
scanning tunnelling (force) microscope

transmission electron microscope

techniques
scanning electron microscope

optical microscope

eye
n FIGURE 1.2 Range of resolution of scanning electron microscopy compared to other microscopic techniques [2].

vacuum. An (ultra) high vacuum is indispensable for SEMs to avoid scat-

tering of the electrons along their path by residual gas atoms (free path
length), high voltage discharge, and contamination of the electron gun
and other electron optical components. The quality of the required vacuum,
i.e., the pressure in the microscope column, is an important cost factor. It
depends, for instance, on the type of the electron gun that is used. Usually,
more than one type of vacuum pump is employed to attain the required vac-
uum. Commonly, a cascade consisting of a mechanical pump and a diffu-
sion pump is utilized to pump down from atmospheric pressure to a basic
vacuum level. Different types and arrangements of pumps are summarized
as follows [4]:
Mechanical pumps consist of a motor-driven rotor that compresses a large
volume of gas into a small volume and thereby increases the gas pressure.
If pressure of the compressed gas is great enough, it can be expelled to the
atmosphere by a unidirectional valve. As the pressure of the chamber
increases, the efficiency of the vacuum pump decreases rapidly, although
it can achieve a vacuum better than 5
105 torr (Torricelli). In diffusion
pumps, vaporized oil circulates from the top part of the pump to the bottom
part. The gas at the top of the pump is transported along with the vaporized
oil to the bottom where it is discharged by the mechanical pump. A heater
component is utilized to vaporize the oil, and coolants cool down the oil so
that it can be used circularly. The diffusion pump can achieve 5
105 torr
rapidly. Diffusion pumps start to work at a pressure of approximately
 1.2 Scanning Electron Microscopy 5

1
102 torr. For that reason, a mechanical pump is used to pump down
the chamber to the pressure at which the diffusion pump can begin to
operate. Such a setup is normally used in SEMs that are operated with a
thermionic tungsten cathode. In turbo pumps, gas molecules are pushed
in a particular direction by the action of rotating blades. The rotor blades
transport molecules from the microscope chamber side to the subsequent
pump in the pumping cascade. Ion pumps can achieve higher vacuum
levels that are necessary for the operation of thermionic lanthanum
hexaboride (LaB6) guns (106 to 107 torr) or field emission guns (109
to 1010 torr).

1.2.3 Electron Beam Generation and Electron Optics

Electrons are generated and accelerated down the column by a so-called
electron gun, and they pass through a set of electromagnetic lenses and
apertures. The electron beam forms a fine probe that impinges the surface
of the specimen in the microscope chamber. The beam itself is scanned
in a rectangular line-by-line manner (“raster”) across the surface of the
sample by means of scan coils that are situated above the objective lens.
The system includes an electromagnetic device (stigmator) to correct astig-
matism as one of the optical aberrations that occur in electron optical
systems.

1.2.3.1 Electron Beam Generation

In Thermionic emission guns, free electrons are produced by heating up a
thermal emitter, namely a tungsten hairpin filament or a pyramidal-shaped
LaB6 crystal. If the temperature is high enough, electrons have sufficient
thermal energy to surmount the work function of the materialevacuum
interface and leave the tungsten cathode. A thermionic electron gun is a
triode system consisting of the cathode (C) connected to a highly negative
potential, the (grounded) anode aperture (A), and the Wehnelt electrode
(W) in between, which has a potential that is hundreds of volts more nega-
tive than the cathode potential (Fig. 1.3D). Therefore, electrons emitted
from the point of the electrically heated cathode are accelerated toward
the anode, and they are strongly influenced by the function of the Wehnelt
electrode. The negatively biased Wehnelt electrode restricts the electron
emission and allows focusing the electrons to a so-called crossover as
they are accelerated to the anode [2]. LaB6 crystals, having a sharp tip as
depicted in Fig. 1.3B, exhibit a much lower work function, give a better
signal-to-noise ratio, and allow smaller beam diameters and, therefore, a
better resolution than tungsten filament cathodes. The lifetime is signifi-
cantly higher. Nevertheless, LaB6 is a very reactive material; the gun
6 CHAPTER 1 Scanning Electron Microscopy, ESEM, and X-ray Microanalysis

n FIGURE 1.3 Scanning electron spectroscopy images of various electron sources: tungsten filament (A), LaB6 emitter (B), tungsten field-emitting tip (C), and
schematic diagrams of a thermionic gun (D) and a field-emission gun (E). From G.H. Michler, Electron Microscopy of Polymers, Springer-Verlag, Heidelberg Berlin,
2008.

vacuum has to be 10e100 times better than that for tungsten cathodes.
LaB6 cathodes are susceptible to thermal shock, so it is important to take
care when heating and cooling.
In field-emission guns (FEGs), free electrons are generated by extraction
from the material, e.g., tungsten, by quantum tunneling. Under ultrahigh
 1.2 Scanning Electron Microscopy 7

vacuum, a material subjected to a sufficiently strong electric field will emit

electrons in the region of maximum field strength. Such a process even
works at room temperature (cold FEG). In Fig. 1.3C, a typical tungsten
cathode with a sharply pointed tip of a radius of 100 nm is depicted. The
function of a field emission gun is shown in Fig. 1.3E: A first anode is posi-
tively biased [several kilovolts (kV)] to the cathode. The extraction voltage
(V1) generates the field strength at the cathode tip to extract electrons. The
second anode accelerates the electrons to the final energy determined by
voltage V2 between the cathode tip and the grounded second anode [2].
The main advantage of FEGs is the generation of a very narrow primary
electron beam, i.e., a very fine electron probe. Another important advan-
tage is the absence of thermal energy spread; the beam is highly monochro-
matic. On the other hand, the beam current is relatively low.
In a thermal FEG, the emitter is heated to a certain temperature. Due to the
reduction of the work function by the strong electric field, the Schottky ef-
fect is responsible for electron emission (Schottky FEG). The work func-
tion of the emitter can be further reduced by covering the tungsten tip
with a thin ZrO layer. This results in a decrease of emission noise and pro-
vides a stable emission current, which is not influenced by contamination
layers. Thus, the Schottky FEG combines high electron yield, stable
beam parameters, and a narrow electron beam. Thus, such a setup is the
choice for instruments that combine high-resolution imaging with analyt-
ical methods.
A comparison of the most important features of electron guns is given in
Table 1.1. It has to be considered that FEG sources require better vacuum,

Table 1.1 Comparison of Features of Different Types of Electron Guns

TE gun

Tungsten LaB6 FE gun SE gun

Electron-source size 15e20 mm 10 mm 5e10 nm 15e20 nm

Brightness (Acm2 rad2) 105 106 108 108
Energy spread (eV) 3e4 2e3 0.3 0.7e1
Lifetime 50 h 500 h Several years 1e2 years
Cathode temperature (K) 2800 1900 300 1800
Current fluctuation (per hour) <1% <2% >10% <1%

Data for an accelerating voltage of 20 kV. TE, thermionic emission gun; FE, field-emission electron gun; SE, Schottky emission electron gun.
From Scanning electron microscope A to Z: basic knowledge for using the SEM, JEOL USA http://www.jeolusa.com/RESOURCES/Electron-Optics/
Documents-Downloads.
8 CHAPTER 1 Scanning Electron Microscopy, ESEM, and X-ray Microanalysis

and they are more expensive than tungsten or LaB6 cathodes. On the other
hand, they provide electron probes of much higher brightness, much lower
energy spread, and a much lower spot size allowing higher resolutions.

1.2.4 Signals, Detectors, and Image Formation

Signal generation in SEM is a result of the interaction between the incident
electron beam and a thin surface layer of the sample, which depends on the
beam energy. The primary electrons (PEs) are charged particles and so they
interact strongly with the electrically charged particles of the atoms in the
sample, i.e., both with negatively charged electron clouds and positively
charged nuclei. The interaction is said to be inelastic if some of the energy
of the PE is lost during the interaction. If no energy is lost, the interaction is
said to be elastic. Because of the interaction of the electron beam with the
specimen, a variety of signals can be collected by appropriate detectors
(Fig. 1.5).
Secondary electrons (SEs) are a result of inelastic scattering of PEs with
weakly bonded electrons of the outer shells of atoms in the whole interaction
volume. They possess a low energy of 3e50 electron volts (eV). Thus, only
SEs from a volume close to the surface escape from the material and can be
collected and amplified by means of appropriate detectors. It has to be
mentioned that SEs can be categorized into SE types I to IV, representing
SEs of different origins. For SE imaging, SE type I originating from the sur-
face of the interaction area itself is considered. SEs are usually exploited to
image the surface topography of a sample. The contrast, in this case, is
largely a function of the tilting angle of the sample surface with respect to
the incident primary electron beam. Moreover, there are so-called edge ef-
fects and shadowing effects, making SE topography images appear in a
very plastic manner.
SEs are usually collected and amplified by means of an Everharte
Thornley detector (Fig. 1.4). The detector is a combination of a scintillator
and a photomultiplier. Low-energy SEs are attracted and collected by a grid
electrode that is positively biased with a voltage of about 200 V. The elec-
trons are accelerated toward the scintillator that is at a voltage of
about þ10 kV. Detected by the scintillating layer, they are transformed
into light and transferred to the photomultiplier (PMT). Here, light causes
emission of photoelectrons that are amplified by electron multiplication.
The result is an electronic signal that is proportional to the amount of
collected SEs.
Backscattered electrons (BSEs) are generated by elastic scattering of PEs
when they interact with sample atoms. Due a much higher energy than SE
 1.2 Scanning Electron Microscopy 9

n FIGURE 1.4 Schematic drawing of the function of an EverharteThornley detector. Redrawn from G.H.
Michler, Electron Microscopy of Polymers, Springer-Verlag, Heidelberg Berlin, 2008; Scanning electron
microscope A to Z: basic knowledge for using the SEM, JEOL USA http://www.jeolusa.com/RESOURCES/
Electron-Optics/Documents-Downloads.

(60%e80% of the energy of the PEs), they are able to escape from deeper
regions of the interaction volume. The signal that is usually detected by a
semiconductor device is strongly dependent on the average atomic number
of the interacting atoms.
Semiconductor BSE detectors consist of two or four semiconductor diodes
which are symmetrically arranged around the opening of the pole piece of
the objective lens. BSEs generate electronehole pairs, and their quantity de-
pends on the energy and the number of the electrons. The output signal is
proportional to the BSEs reaching the detector. Depending on the processing
of the signal outputs from the different diodes, it is possible to generate both
topography-sensitive and material-specific signals [2].
Signals may also be created by processes in which electrons are knocked
out of (inner) electron shells of an atom due to the impact of PEs. Atoms
enter an excited energy state. The empty electron orbitals have to be filled
by an electron from outer shells. The energy can be released through the
emission of a photon. Alternatively, in X-ray emission, energy is released
through the emission of an Auger electron (AE). As AEs possess relatively
low energies, similarly to SEs, they can only escape from a depth of a few
nanometers. If the above mentioned vacant electron state is an outer state
(outer electron shells), the energy difference is smaller, and the emitted
photon will be in the visible wavelength range. The cathodoluminescence
(CL) phenomenon can be exploited as a signal for image formation
(Fig. 1.5).
Because the incident electrons are able to penetrate into the sample volume,
interactions between the PEs and atoms of the material take place within a
certain interaction volume (Fig. 1.6). The penetration depth and, hence,
10 CHAPTER 1 Scanning Electron Microscopy, ESEM, and X-ray Microanalysis

n FIGURE 1.5 Schematic drawing of the interactions between primary electrons (PEs) of the incident
electron probe and an atom. From G.H. Michler, Electron Microscopy of Polymers, Springer-Verlag,
Heidelberg Berlin, 2008.

the size of the bulblike interaction volume strongly depend on the energy of
the PEs that is defined by the accelerating voltage as well as on the material
properties (composition, average atomic number). The complex character of
the interaction, the energies and escape depths of the detectable signals, and
the consequences concerning the resolution of SEM imaging are described in
detail in a book by Reimer [6].
There are different types of contrast mechanisms that are exploited in SEM.
Of course, the intensity of signals strongly depends on the average atomic
number of the elements that are at hand inside the interaction volume,
causing a material contrast. Similarly, signals can strongly depend on the
orientation of crystalline structures (grain structure, texture of the crystal-
line material). Moreover, the signal intensity strongly depends on topo-
graphic features of the sample surface. The important mechanisms of
topographic contrast formation are the following:
Topographic contrast is obtained because the intensity of SE emission is a
function of the tilt angle between the incident electron beam and the sample
surface area under investigation. The smaller the angle, the bigger is the
streaking path for the interaction of PEs and the surface layer from which
SEs and BSEs are able to escape, and the bigger is the cross-sectional
 1.2 Scanning Electron Microscopy 11

n FIGURE 1.6 Schematic of the interaction volume with the representation of the origin of the different
signals. Redrawn from RÖDER, lecture script Martin Luther University HalleeWittenberg.

area of the interaction volume. Thus, the signal intensity increases with
smaller incident angles (Fig. 1.7A).
If the detector is placed laterally with respect to the sample, contrast can be
obtained by a shadowing effect. Electrons that are escaping from areas that
are facing toward the detector are more easily detected than electrons that
emerge from surfaces that are far from the detector or that are facing to

n FIGURE 1.7 Visualization of two effects of topographic contrast generation: (A) tilt effect, (B) edge
effect. From L. Reimer, Scanning Electron Microscopy: Physics of Image Formation and Microanalysis,
Springer Science & Business Media, 1998.
12 CHAPTER 1 Scanning Electron Microscopy, ESEM, and X-ray Microanalysis

the opposite direction. Thus, object areas that are turned away from the
detector appear darker than areas faced toward the detector. For that reason,
it is essential to know the position of the detector with respect to the direction
of observation!
Edge effect is similar to the abovementioned tilt effect. It is more pro-
nounced in samples consisting of lighter elements. Here, sharp edges that
stand out of smooth areas or between tilted areas appear exceptionally bright.
This effect is caused by the steep shoulders of an extreme tilt angle. On the
other hand, pits and reentrant edges (notches) appear dark (Fig. 1.7B).
Roughness-induced contrast is a consequence of the edge effect: A surface
having (microscopic) roughness will appear brighter than a (microscopi-
cally) smooth surface of the same material due to the multitude of small
edges with high signal intensity.

1.2.5 Sample Preparation

SEM sample preparation requires, first, a clean desk, if possible in a dust-
protected environment, and appropriate tools. One should always use clean
blades, tweezers, etc., that can be defatted, e.g., using acetone. Contamina-
tion by humidity, dust, fingerprints, rinsing, or etching agents must be
avoided. To protect the sample and with respect to the high vacuum in
the SEM gloves are recommended. Delicate samples should be stored in
a desiccator or under vacuum.
Sample preparation can be quite simple and straightforward (powders, free-
standing surfaces, fracture surfaces). Very often, preparation means manip-
ulating the sample to expose the region of interest (defects, internal
structures, morphology, or texture of a heterogeneous material). Very often,
it is necessary to produce cross sections of a bulk sample in a defined spatial
direction. Biological and biomedical samples require special treatments for
the conservation of structures [8]. More complex samples, such as hybrid
packages or microelectronic devices, require the application of a sequence
of sophisticated multistep procedures including the localization of a defect
inside packed units (e.g., using lock-in thermography [9]), chemical removal
of encapsulations, and/or mechanical manipulation using milling machines
followed by laser and/or ion beam milling procedures of increasing precision
and conservation of original structures. A collection of basic steps follows.

1.2.5.1 Mounting
Samples are mounted on SEM stubs of a size and material specified by the
instrument supplier. Note that the sample must not exceed the size given by
 1.2 Scanning Electron Microscopy 13

the chamber size or lock (door) size. The height of the sample is crucial: It
defines the possible range of working distance that can be achieved. The
sample itself can be clamped mechanically, or it can be glued by a special
(conductive, vacuum compatible) glue or by means of double-sided adhe-
sive SEM pads. The samples should be fixed well to avoid any movement
under electron beam irradiation due to heating or charging. Smaller bulk
samples and powders can be mounted and observed directly. Small amounts
of a powder, for example, can be pressed slightly on double-sided adhesive
pad, and residual powder can be removed carefully, e.g., by compressed air.
The material should be fixed in a manner that a contamination of the micro-
scope column is avoided. Special care should be taken with magnetic items/
particles.

1.2.5.2 Fracture Surfaces

Fracture surfaces are found because of a catastrophic failure of a material or
a part or after mechanical testing of an engineering sample. In this case, the
damage or fracture analyses in the SEM are performed directly. Very often,
artificial fracture surfaces are produced to gain insight into a bulk material
or to produce a representative cross section of a sample. The technique de-
pends on the sample properties: A brittle material can be broken, for
instance, by means of a hammer. More ductile or elastic materials like
many polymers and rubbers must be hardened; the most common way is
to freeze the samples well below their glass transition temperature by means
of liquid nitrogen. Thus, brittle fracture can be achieved.

1.2.5.3 Microtomed Sections and Cross Sections

A more elegant way to produce cross sections of a sample is to produce
thin slices (sections) of a thickness of several hundred nanometers to
some tens of microns using (ultra) microtomes or cryo-ultramicrotomes
(for soft materials). The procedures are similar to those applied for light
optical microscopy or transmission electron microscopy (TEM). Sections
can be placed, for instance, on standard SEM stubs for SEM observation.
Alternatively, sections can be fixed on perforated TEM copper grids. In
this case, the observation of thin sections of optimum thickness in the
SEM can improve the resolution especially for BSE imaging, because
signals from very deep regions of the interaction volume (and, therefore,
relatively broad escape areas) are excluded. Such a technique is applied to
operate an SEM as a kind of scanning transmission microscope. Neverthe-
less, it is more common to use the microtomed block face for SEM
investigations.
14 CHAPTER 1 Scanning Electron Microscopy, ESEM, and X-ray Microanalysis

1.2.5.4 Target Preparation

As mentioned earlier, it might become necessary to prepare SEM samples
from a defined region of a complex part or component. Moreover, such
samples might require a quite delicate handling because the sample is
unique and not easy to reproduce. In such a case, the following techniques
(or combinations) can be applied.
A relatively rough preparation can be done by sawing and mechanical mill-
ing. For the latter, instruments are available that allow mechanical manipu-
lation under light optical control so that the preparation can be very exact
within some tens of micrometers. Very smooth surfaces can be achieved
by mechanical polishing. Such surfaces are suitable for higher-resolution
images of samples showing material contrast or for analytical SEM. Ion
milling is applied to manufacture extremely smooth surfaces or to thin
down samples to electron transparency.

1.2.5.5 Focused Ion Beam

This method is probably one of the most elegant ways to produce smooth
and ultrathin samples for electron microscopic observation [10]. SEM and
TEM samples can be prepared with excellent conservation of structures,
especially interfaces between different classes of materials. The method
is especially suitable for the preparation of microscopic regions of interest
(e.g., a single transistor in a semiconductor chip): The field of operation is
observed using an SEM that is incorporated into the focused ion beam
(FIB) instrument. The design of an FIB instrument is similar to that of
the SEM. In the case of the FIB, a beam of gallium ions is emitted from
a liquid-metal ion source and is focused by electrostatic lenses. The spot
size is in the range of a few nanometers. The impinging ion beam is
removing atoms from the sample surface (sputter process). Because the
sputter process acts very locally, very fine trenches can be prepared. An
example is given in Fig. 1.8A: On two opposite sides of a region of interest,
trenches are cut by a line-by-line removal of the sample material. By that
process, a so-called lamella is thinned down to the desired thickness (usu-
ally to electron transparency, which is some tens to some hundreds of
nanometers, depending on the material). Subsequently, the ligaments of
the lamella are cut, and the sample is transferred to a TEM or high-
resolution SEM (Fig. 1.8B).
Whereas FIB technique is the gold standard for many materials and systems
all across the areas of electronics, metallurgy, petrology, ceramics, and so
on, beam-sensitive materials such as many polymers and biological tissues
 1.2 Scanning Electron Microscopy 15

n FIGURE 1.8 Example for the preparation of samples using FIB technology. (A) Sample surface showing the trenches dug at each side of the lamellae; and
(B) silica nanoparticles embedded in a polymer matrix become visible within the thin and smooth lamellae.

still are delicate to handle by FIB. In this case, ultramicrotomy might be an

alternative.

1.2.5.6 Drying
For all investigations using a standard SEM setup (i.e., high vacuum in the
microscope chamber), it is necessary to use a dry sample. This means, in the
first sense, that water has to be removed completely. This might cause
problems, because original structures may be disturbed or may collapse
during drying procedures. There are well-established procedures for
water-containing samples, such as most simple techniques like using an
ascending alcohol series or acetone baths, or instrumented methods such
as freeze-drying or critical-point drying. It should be mentioned that the
same holds for residual solvents, fats, oils, and surfactants that are contained
inside or present at the surface of a sample. Similarly, porous samples may
contain large amounts of gas molecules that should be removed by
degassing.

1.2.5.7 Conductive Coating

All investigations using a standard SEM setup (i.e., high vacuum in the
microscope chamber) require a conductive sample or sample surface.
For nonconductive samples, such as polymers, biological materials,
ceramics, and many more, a conductive coating is obligatory to avoid
charging of the sample that would make SEM observation impossible.
The selection of the proper coating technique is crucial: On one hand,
16 CHAPTER 1 Scanning Electron Microscopy, ESEM, and X-ray Microanalysis

metal coatings increase the SE yield and, therefore, may improve the topo-
graphic contrast of samples consisting of light elements. On the other
hand, such coatings will cover any material contrast and overlay X-ray
spectra so that elemental analyses become difficult. In the latter case,
conductive coating should be realized by the vacuum deposition of a
thin carbon layer. Typical coatings consist of 5e20 nm layers of carbon,
gold, platinum, or chromium and can be produced by means of a sputter
coater (Au) or by vacuum evaporation of C, Pt, Cr, or Au. Combinations
of different materials are possible. It is noteworthy that all deposition
layers exhibit a more or less pronounced structure. Gold coatings are rela-
tively coarse and should not be used for high-resolution SEM imaging in
which Pt and Cr coatings are favorable.

1.2.6 Examples
A number of examples should illustrate the main aspects of contrast gener-
ation and interpretation. They represent different ways of preparation and
different classes of materials. Fig. 1.9 shows a bunch of glass fibers as
they are commonly used to reinforce polymers. The polymeric sample
was ashed at 700 C, and the residue was placed on a conductive adhesive
pad. Because energy-dispersive X-ray microanalysis (EDX) investigations
were required, the sample was carbon coated (evaporation, approximately
15-nm layer thickness). Images were taken using a standard SEM
equipped with a tungsten thermionic gun. SEs were recorded using an
EverharteThornley detector (ETD). To achieve adequate depth of focus,
the working distance was relatively high (39 mm).

n FIGURE 1.9 Scanning electron microscopy Images of glass fibers, secondary electron mode at 15 kV: (A) overview image, (B) detailed image. Note the
excellent depth of focus and the almost stereoscopic appearance of the images. The edge effect is obvious (detector is placed on the right-hand side).
 1.2 Scanning Electron Microscopy 17

n FIGURE 1.10 Surfaces of urinary stents after explantation. (A) Mineral crust, (B) microorganisms and salt crystals. Secondary electron mode, 15 kV. Note that
the rough organic residues appear much brighter than the flat surface layer.

Fig. 1.10 presents images from untreated surfaces of urinary stents that were
explanted from the human body after failure (incrustation). Samples were
air-dried, carbon coated, and investigated under conditions similar to those
described previously. Again, the preparation is reduced to the very basic
steps of drying, mounting, and conductive coating.
The morphology of a stent is revealed in Fig. 1.11. The pair of pictures
shows the same cross section produced by means of a microtome equipped
with a metal blade and operated at room temperature. The picture on the left-
hand side is recorded using SEs detected by an ETD, whereas the right-hand
side picture is taken using the BSE signal collected by a semiconductor
detector working in the material-contrast mode. Due to carbon coating, the

n FIGURE 1.11 Cross section of a polymeric stent containing X-ray opacifier particles. (A) Secondary electron image, (B) backscattered electron material-contrast
image. In picture (A), the topographic contribution allows identification of the polymeric matrix material, whereas the material-contrast picture (B) highlights
the component consisting of elements with higher atomic number.
18 CHAPTER 1 Scanning Electron Microscopy, ESEM, and X-ray Microanalysis

n FIGURE 1.12 Imaging of carbon nanotubes (CNTs) using a field-emission gun scanning electron microscopy. (A) As-received nanotube powder,
(B) cryofracture surface of a polymer containing CNT. Note the high-resolution reproduction of the nanotubes.

conductive layer is not disturbing the material-contrast image. The bright

spots represent the X-ray opacifier that was added to the polymer for the pur-
pose of X-ray-supported positioning in the body.
The next example shall be used to demonstrate the difference between a stan-
dard SEM equipped with a thermionic electron gun and an FEG instrument.
The sample in Fig. 1.12A is an as-received carbon nanotube (CNT) powder
loosely mounted on an SEM stub covered with a conductive double-sided
adhesive tape. Conductive coating is not necessary because the material itself
is conductive. Fig. 1.12B reproduces a cryofracture surface of a polymeric
material with embedded CNT and additional filler particles. The combination
of the advantages of the FEG source and the absence of any coating result in
high-resolution images of the CNT. For the right-hand side picture, a very low
working distance of 1.3 mm was chosen. Accelerating voltage is 5 kV, and
SEs were detected using ETD.

1.3 ENERGY DISPERSIVE X-RAY MICROANALYSIS

1.3.1 Signal Generation and Detection
Characteristic X-rays originate from a process in which electrons are
knocked out of (inner) electron shells of an atom due to the impact of
PEs. As a result, the atom enters an excited energy state. Subsequently,
the empty electron orbital is filled by an electron jumping into the gap
from outer shells. The energy can be released through the emission of a
photon. Because the energy difference depends on the electron configuration
of the atom, the emitted characteristic X-ray is a “finger print” of the atom
 1.3 Energy Dispersive X-ray Microanalysis 19

that takes part in the interaction. The collection of characteristic X-ray

spectra allows the analysis of the elemental composition of the sample at
the spot of investigation [e.g., energy-dispersive X-ray microanalysis
(EDX)]. By the acquisition of characteristic X-ray spectra at different spots
in a line-by-line manner (the “raster” principle), so-called mappings repre-
senting different elements can be recorded. It has to be mentioned that the
point resolution of X-ray mappings is much lower than that of SE images
because X-ray signals escape from much deeper regions of the interaction
volume (cp. Fig. 1.5B).
The collection of X-ray quanta requires special detectors. Generally, X-ray
analyses can be performed in two different ways: On one hand, the so-
called wavelength-dispersive X-ray microanalysis (WDX) was the original
method for elemental analysis by electron-induced X-ray emission. This rela-
tively time-consuming method is employed when high spectral resolution is
required (5e20 eV), or element concentrations of less than 0.1 wt% have to
be detected. On the other hand, energy-dispersive X-ray microanalysis
(EDX) is able to acquire spectra much faster. The sensibility of the method
is lower; depending on the atomic number of the element, the concentration
must be at least 0.1 to 1 wt%. Today, most of the instruments are equipped
with EDX detectors that allow element identification, quantitative X-ray
analyses, and EDX mappings within economical time frames. More detailed
information on detector design, signal processing, quantitative analyses,
sensitivities, and limitations should be found in a number of text books, e.g.,
Refs. [2,7]. A comparison of advantages of EDX versus WDX is presented
in Ref. [3].

1.3.2 Examples
The usefulness of EDX analyses is illustrated by only two examples.
Fig. 1.13 gives a typical result that is received by the SEM investigation
of bone. The sample is prepared following classical histological steps, as
there are, fixation, dehydration, embedding and polishing. Such a very
smooth surface can be imaged perfectly by means of the BSE material-
contrast signal. The gray levels that are clearly distinguishable in
Fig. 1.13A represent different levels of the mineralization of bone. The
brighter the image spot, the higher is the concentration of the bone mineral.
The composition of bone mineral can be derived from the EDX spectrum
that was recorded from the whole sample region: As one would expect,
the main constituents are phosphorus, calcium, and oxygen. The carbon
peak is caused by several contributions: the organic component of bone
itself, the polymeric embedding material, and the conductive carbon coating.
Accelerating voltage is 15 kV.
20 CHAPTER 1 Scanning Electron Microscopy, ESEM, and X-ray Microanalysis

(A) (B)
5000 Ca

P
4000

3000
Ca
C

2000 P

1000
O Ca

Na
0 keV
0 10.00

n FIGURE 1.13 Scanning electron microscopy analysis of a polished bone sample. (A) Backscattered electron material-contrast image revealing areas of different
degrees of mineralization; (B) EDX spectrum proving the presence of the elements C, O, Na, P, and Ca.

Fig. 1.14 illustrates the opportunity to generate images of the element distri-
bution within a selected area of the sample. The overall situation can be seen
in Fig. 1.14A: The cross section of a silicone implant reveals the interface
between a plaque and the base material. The problem was to show the nature
of the incrustation and to give evidence for the hypothesis that the plaque is
directly bound to the base material. After making sure that the base material
is characterized by a sharp Si peak and that the crust mainly consists of the
elements Ca and P, the elemental mapping in the region of interest was
limited to these three elements. As a result, one observes a sharp interphase
also in the EDX mapping, allowing the localization of the significant phases.

1.4 ENVIRONMENTAL SCANNING ELECTRON

MICROSCOPY
One of the outstanding achievements in SEM was the invention of instru-
ments that allow variable pressures (lower vacuum or even atmospheric
conditions) in the microscope sample chamber while maintaining ultrahigh
vacuum that is essential for beam generation in the column. The history of
the invention, the technical details, and the wide field of applications are
discussed in a number of publications [11e19]. It is noteworthy that the
original term “environmental scanning electron microscopy” or “ESEM”
is often used simultaneously with the terms “variable pressure SEM”
(“VP-SEM”) or “low vacuum SEM” (“LV-SEM”), depending on the instru-
ment manufacturer and different definitions of the range of pressure
variation.
 1.4 Environmental Scanning Electron Microscopy 21

n FIGURE 1.14 Secondary electron (SE) image and energy dispersive X-ray microanalysis mappings of the interface between a mineral incrustation and the
base material of a silicone stent; (A) SE image showing the region of interest; (BeD) element distribution of the elements Si (B), Ca (C), and P (D).

1.4.1 Principle and Advantages

The main trick behind ESEM is the separation of the two different pressure
levels in the microscope chamber in which the sensitive sample is posi-
tioned and the column in which ultrahigh vacuum is required. For that pur-
pose, one can imagine a pinhole small enough to hinder gas molecules to
pass but large enough for the very thin electron beam. If this is done repeat-
edly in a cascade in which the few gas molecules are removed by vacuum
pumps, one gets a so-called pressure-limiting aperture. Leaving the final
22 CHAPTER 1 Scanning Electron Microscopy, ESEM, and X-ray Microanalysis

aperture, the electron beam is subject to intensive scattering processes in the

low-vacuum chamber, depending on the path length (i.e., working distance)
and the nature and pressure of the gas inside the chamber. The resulting
“skirt effect” negatively influences the signal-to-noise ratio, because the
relation between finely focused electrons and scattered primary electrons
that contribute to the image formation is getting worse the more scattering
appears. Nevertheless, there are striking advantages:
1. Under certain conditions (optimum water vapor pressure and tempera-
ture), it is possible to investigate wet (nondehydrated) samples.
2. Because ionization of the residual gas is taking place, charge can be
removed from the surface of nonconducting samples; conductive
coating becomes obsolete.
3. Alternative detection routes are possible (gaseous secondary electron
detector).
The first paragraph opened up a new field of SEM applications in biology,
medicine, food and cosmetic industry, paint and concrete science, and so
on. Together with the fact that conductive coating was not needed, the
ESEM became a true “lab in the SEM.” The design of a number of
in situ experiments became possible. Thirdly, a new detection route was
at hand because the conventional ETD did not work under low-vacuum
conditions.

1.4.2 Instrumentation
Besides the cascade of pressure stages, ESEM technology requires a special
vacuum pump setup. Additionally, gases such as water vapor or nitrogen
can be fed in to the chamber by a valve dosage system. ESEMs are usually
equipped with a cooling stage because wet samples have to be cooled down
to enable “wet” imaging. The microscope column itself is very much the
same as for conventional SEM. Nevertheless, it is recommended to look
for an FEG electron source rather than a thermionic gun because both res-
olution and the signal-to-noise ratio that is reduced by skirt effect can be
improved significantly. SE detection usually works with the gaseous SE
detector, using the ionized gas itself as a signal amplification cascade.
BSEs are usually detected by means of a semiconductor device placed right
below the final aperture.

1.4.3 Examples
The following pictures are used to illustrate the main features of ESEM
investigation. The first figure (Fig. 1.15) shows electrospun polymeric
nanofibers. The diameter of the fibers is in the range of 100 nm to some
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