Engineering Materials: For Further Volumes

Engineering Materials
For further volumes:

http://www.springer.com/series/4288
Vikas Mittal Nadejda B. Matsko

Analytical Imaging
Techniques for Soft Matter
Characterization
123
Vikas Mittal Nadejda B. Matsko
Chemical Engineering Department Institute for Electron Microscopy
The Petroleum Institute and Fine Structure Research
Room Number 2204 Technical University of Graz
Abu Dhabi Graz
UAE Austria
ISSN 1612-1317 ISSN 1868-1212 (electronic)

ISBN 978-3-642-30399-9 ISBN 978-3-642-30400-2 (eBook)
DOI 10.1007/978-3-642-30400-2
Springer Heidelberg New York Dordrecht London
Library of Congress Control Number: 2012939481
Springer-Verlag Berlin Heidelberg 2012

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Preface
This book aims to describe the microscopic characterization of the soft matter in
the light of new advances acquired in the science of microscopy techniques such as
AFM; SEM; TEM, etc. It does not focus on the traditional information on the
microscopy methods as well as systems already present in different books, but
intends to answer more fundamental questions associated with commercially
important systems by using new advances in microscopy. Such questions are
generally not answered by other techniques. The contents of this book also reflect
this as the chapters are not based on describing only the material systems, but are
also based on answering the problems or questions arising in their characterization.
Both qualitative as well as quantitative analysis using such microscopic techniques
are discussed. Moreover, efforts have been made to provide a broader reach as
discussions on both polymers as well as biological matter have been included as
different sections. Such a text with comprehensive overview of the various char-
acterization possibilities using microscopy methods can serve as a valuable ref-
erence for microscopy experts as well as non-experts alike. We are deeply
indebted to Dr. Anton Efimov, and Dr. Victoria Klang for providing perfect cryo
AFM and cryo TEM projects. We are very grateful to the team of FELMI-ZFE,
especially to Ilse Letofsky-Papst, Michaela Albu Franz Schmidt and Werner
Grogger for the experimental support and fruitful discussions. Our special thanks
go to Ferdinand Hofer and Martin Mueller for their support during this work. We
would like to also thank Prof. Claudia Valenta, Prof. Otto Glatter, Prof. Andreas
Zimmer, and Dr. Nada Znidarsic who provided us interesting samples. Co-oper-
ation with Austrian Cooperative Research (ACR) in Vienna, ETH Zurich, and FFG
foundation, Austria is highly appreciated.
Vikas Mittal
Nadejda B. Matsko
v
Contents
Part I Introduction
1 Introduction to Microscopy Techniques. . . . . . . ............. 3

1.1 Which is More Valid: Information Obtained
by Eyes or by Hand? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.2 Transmission Electron Microscopy . . . . . . . . . . . . . . . . . . . . 5
1.2.1 Analytical TEM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.3 Scanning Electron Microscopy (SEM). . . . . . . . . . . . . . . . . . 7
1.4 Atomic Force Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . 7
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Part II Biological Related (Hydrated) Matter
2 Visualization of OrganicInorganic Nanostructures in Liquid . .. 13

2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 13
2.2 Analytical Techniques Employed for the Characterisation
of Colloidal Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 15
2.3 Transmission Electron Microscopy: Experimental Setup,
Sample Preparation and Potential Artefacts . . . . . . . . . . . . .. 15
2.4 Cryogenic Transmission Electron Microscopy (Cryo TEM):
Experimental Setup, Sample Preparation
and Potential Artefacts . . . . . . . . . . . . . . . . . . . . . . . . . . .. 16
2.5 Laser Light Scattering Versus Electron Microscopy . . . . . . .. 20
2.6 Freeze-Etching and Freeze-Fracturing of Nanoemulsions
for Transmission Electron Microscopy (FFTEM):
Experimental Setup, Sample Preparation
and Potential Artefacts . . . . . . . . . . . . . . . . . . . . . . . . . . .. 23
2.7 Scanning Electron Microscopy, Cryo SEM
and Freeze-Fracture SEM: Experimental Setup,
Sample Preparation and Potential Artefacts . . . . . . . . . . . . .. 25
vii
viii Contents
2.8 Recent Advances: Cryo Analytical TEM (cryo ATEM). . . . . . 27

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
3 Macromolecular Distributions in Biological

Organisms In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 31
3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 31
3.2 AFM Tuning Parameters Used for the Imaging
of the Epoxy Embedded Biological Samples . . . . . . . . . . . .. 34
3.3 Dependence of the AFM Phase and Topographical Contrast
on the Integrity of Cellular Protein Molecules . . . . . . . . . . .. 35
3.4 Correlative AFM/TEM Analysis of the Protein Preservation
in the Samples, Prepared in Accordance with Different
Freeze-Substitution Protocols . . . . . . . . . . . . . . . . . . . . . . .. 38
3.5 Identification of the Cell Constituents in AFM Phase
Image Using AFM and TEM Complementary
Couples of Images . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 41
3.6 Interpretation of the TEM Images Obtained from the Epoxy
Fixed Sample Using Complementary AFM Data . . . . . . . . . . 43
3.7 Difficulties in the AFM Image Interpretation . . . . . . . . . . . . . 45
3.8 Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
4 Structure of the Biological Membrane (Detection

of the Membrane Components In Vivo). . . . . . . . . . . ......... 49
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . ......... 49
4.2 Cellular Membrane: Components and Functions. . ......... 49
4.3 Practical Aspects of Sample Preparation, Sources
of Error and Common Artefacts . . . . . . . . . . . . . . . . . . . . . . 50
4.4 Plasma Membrane by TEM . . . . . . . . . . . . . . . . . . . . . . . . . 52
4.5 Plasma Membrane by AFM . . . . . . . . . . . . . . . . . . . . . . . . . 58
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
5 Structural and Analytical Chemical Analysis of the

OrganicInorganic Components in Biomineralized Tissue . . . ... 61
5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... 61
5.2 General Mechanism of Biomineralization . . . . . . . . . . . . ... 62
5.3 High Resolution Microscopical and Analytical Techniques
Used for the Investigation of the Mineral Phase . . . . . . . . . . . 63
5.4 Correlative AFMTEM . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
5.5 Sample Preparation Procedure . . . . . . . . . . . . . . . . . . . . . . . 71
5.6 Polysaccharides-Protein Filaments . . . . . . . . . . . . . . . . . . . . 73
5.7 Analytical TEM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
Contents ix
6 Cellular Dynamics (Protein Transport,

Mineralization In vivo) . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 77
6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 77
6.2 Detection of the Macromolecular Content of a Cell
by AFM and its Interpretation Using Complementary
AFMTEM Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . .... 78
6.3 Spherules Involved in Elaboration of Crustacean Cuticle
During the Molt Cycle: A Correlative TEMAFM Study .... 81
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 83
7 Tomography of the Hydrated Materials . . . . . . . . . . . . . . . . . . . 85

7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
7.2 Cryo TEM Tilt Serious Based Tomography . . . . . . . . . . . . . . 86
7.3 Cryo AFM Serial Section Tomography . . . . . . . . . . . . . . . . . 87
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
Part III Polymer-Based Matter
8 Morphology in OrganicInorganic Composites . . . . . . . . . . . . . . 97

8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
8.2 What is the Morphology of the Composite Material Containing
Low Aspect Ratio/Platy/Fibrous Filler Particles? . . . . . . . . . . 98
8.3 Do the Filler Substrates with Different Charge Densities
Affect the Morphology? . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
8.4 Do Different Filler Surface Modifications
Affect Morphology? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
8.5 What is the State of Filler Alignment in the Composite?. . . . . 108
8.6 How is the Morphology Developed When Polymer
is Dispersed in Polymer? . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
8.7 What Additional Information Can Be Generated About
Composites on Combining Different Techniques?. . . . . . . . . . 111
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
9 Interface Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 115

9.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 115
9.2 What is the Impact of Small Amount of Compatibilizer
on the Polymer Morphology? . . . . . . . . . . . . . . . . . . . . .... 115
9.3 What is the Composition of Interface in
Polymer/Compatibilizer/Filler Nanocomposite? . . . . . . . .... 119
9.4 What are the Morphological Features of Polymer
Blends Stabilized by Nanoparticles? . . . . . . . . . . . . . . . .... 120
9.5 How Does the Interfacial Morphology in Blends Change
as a Function of Different Compositions? . . . . . . . . . . . .... 122
x Contents
9.6 How Does the Morphology in the Blends Evolve

When Specific Processing Conditions are Used? . ......... 124
9.7 How Does the Morphology in the Blends Seem
in 3D Microscopic Model? . . . . . . . . . . . . . . . . ......... 125
9.8 What is the Morphology in a Block Copolymer? . ......... 125
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......... 125
10 Surface and Volume Characterization . . . . . . . . . . . . . . . . . . ... 127

10.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... 127
10.2 What is the Overall Morphology in the Volume
of the Sample? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... 127
10.3 Which Defects in the Volume of the Samples can
be Detected? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... 132
10.4 How Does the Surface Morphology Change as a Function
of Synthesis Equipment? . . . . . . . . . . . . . . . . . . . . . . . . ... 135
10.5 How Does the Surface Morphology of Particles Change
as a Function of Reaction Conditions? . . . . . . . . . . . . . . ... 135
10.6 What is the Surface and Volume Morphology in Particle
Decorated Particles and Hollow Inorganic Particles?. . . . . ... 142
10.7 What is the Volume Morphology in a Polymer Monolith
Formed by Association of Primary Particles? . . . . . . . . . . ... 143
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... 144
11 Confirmation of Surface Reactions . . . . . . . . . . . . . . ......... 147

11.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . ......... 147
11.2 Has the Reaction on Clay Surface Taken Place? . ......... 147
11.3 How can the Visual Characterization Confirm
the Surface Reaction? . . . . . . . . . . . . . . . . . . . . ......... 149
11.4 How can EDAX and EELS Quantify the Success
of Surface Reactions? . . . . . . . . . . . . . . . . . . . . ......... 158
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......... 161
12 Interactions Between Components. . . . . . . . . . . . . . . . . . . . . . .. 163

12.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 163
12.2 Is There an Interaction Between the Polymer Chains
and the Filler Surface? . . . . . . . . . . . . . . . . . . . . . . . . . . .. 163
12.3 What is the Interaction of the Surface of Particles
with External Species? . . . . . . . . . . . . . . . . . . . . . . . . . . .. 169
12.4 How Does the Surface Interact with Charged Substrate,
or Stimulants Like Temperature, Salt, Sonication etc.? . . . . .. 170
12.5 What is the Interaction Between Polymer Particles Grafted
by Another Polymer Layer? . . . . . . . . . . . . . . . . . . . . . . . .. 172
12.6 How is One Inorganic Species Interacting with the Surface
of the Other (Decoration of the Surface)? . . . . . . . . . . . . . .. 173
Contents xi
12.7 How Does the Solvent Interact with the Morphology? . . . . . . 179
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
13 Nano to Micro and Macro Characterization . . . . . . . . . . . . . ... 183

13.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... 183
13.2 What is the Morphology of the Porous Polymer Network? ... 183
13.3 What is the Morphology of Polymeric Films
on Substrates? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... 189
13.4 What is the Morphology of an Inorganic
Macroporous Network? . . . . . . . . . . . . . . . . . . . . . . . . . ... 191
13.5 What is the Photonic Crystal Morphology? . . . . . . . . . . . ... 193
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... 197
Part I
Introduction
Chapter 1
Introduction to Microscopy Techniques
1.1 Which is More Valid: Information Obtained by Eyes

or by Hand?
The answer to this question is quite obvious: in order to obtain the comprehensive
impression about surrounding objects, one needs both of these sensation systems.
Importantly, the information obtained visually and tactilely is not the same since
different detection mechanisms are applied. This example provides a perception of
diversity and similarity between detection mechanisms of three main high reso-
lution microscopy techniques [transmission electron microscopy (TEM), scanning
electron microscopy (SEM) and atomic force microscopy (AFM)] and human
visual and tactile sensation systems. In TEM, the image contrast is formed by
sufficiently scattering structures that are present in the 20 to 90 nm thick volume of
the section (Fig. 1.1). The technique has a highest instrumentation resolution
(down to 0.5 ), and provides both ultrastructural analysis of the object along with
its chemical composition (analytical TEM). However, TEM has a certain limita-
tions for the analysis of soft matter. Most of biomacromolecules (proteins, poly-
saccharides, nucleic acids) as well as polymer/copolymer chains mainly consist of
light elements (C, H, O, N), which scatter the incident electrons rather weakly and
consequently can be detected on TEM micrographs only as a greyish background.
Staining of the sample can considerably improve the image contrast, but the heavy
metal salts, which are used for such purpose, have a certain size and can penetrate
deep into sample only when macromolecule/chain matrix is relatively disengaged.
Therefore, in TEM image even after staining one does not recognize the whole
ultrastructure, but only those cellular/polymer components which can be reached
by the staining agents and which can react with the latter.
In contrast to TEM, an AFM is a surface oriented technique, which allows one
to get a topographical profile of the sample along with its mechanical character-
istics. In case when the goal is to investigate the bulk of the soft material (e.g.,
polymer), sample has to be sectioned first. A pronounced topographical profile,
V. Mittal and N. B. Matsko, Analytical Imaging Techniques for Soft Matter 3

Characterization, Engineering Materials, DOI: 10.1007/978-3-642-30400-2_1,
4 1 Introduction to Microscopy Techniques
Fig. 1.1 The principle of image contrast formation by a TEM, b SEM and c AFM. Upper line of
images represents a rubber-silica-carbon black nano composite, which has been investigated
using TEM, SEM and AFM respectively
which is detected on the surface of the sample block face, is due to the relaxation
of the tension inside the polymer block during or immediately after the sectioning
process. Such tension most probably results from an electrostatic repulsion
between side groups of (bio-)polymer chains, which are not cross-linked during
polymerization. AFM also detects phase shifts, which in case of biological samples
can be attributed to the different density of the pure embedding resin and the
copolymerized cell components, surrounded by the resin. For artificially synthe-
sized polymers, phase contrast usually shows chain and crystalline order, structure
and distribution of the highly oriented molecules in fibers and films etc. The main
drawback of AFM is that the particular information about chemical composition of
the sample is missing. Usually SEM is used for the analysis of samples with high
surface corrugation, and for the investigation of the large sample areas. Such
information cannot be obtained by TEM or AFM. SEM images also have an
impact from surface topography, which is especially important for the samples
with complex 3D morphology. Currently many scanning electron microscopes
have already implemented WDX detectors which also allow one to gain chemical
information of the samples. The main drawback of SEM is a lack of instrumen-
tation resolution as compared to TEM or AFM, and also a beam damage issue.
In this book, some examples, where a synergetic use of above described
techniques may bring up a new, more comprehensive understanding of soft
materials, are presented. The preservation of structural nativity during sample
preparation as well during microscopy study would be emphasized across the
1.1 Which is More Valid: Information Obtained by Eyes or by Hand? 5
whole book. Some aspects of the image interpretation procedure which is not so
trivial especially for the low contrast micrographs of soft materials would also be
included. And, last but not the least, the book also provides a description of
absolutely new instrumentation (as cryo AFM for cryo 3D tomography) and
methodological approaches (like EFTEM topography measurements of negatively
stained organic materials) which will be of interest for specialists in fields of smart
material development as well as microscopy analysis.
1.2 Transmission Electron Microscopy
Conventional transmission electron microscopes [1] are electron optical instru-

ments analogous to light microscopes. However, the specimen is not illuminated by
light, but by an electron beam (Fig. 1.1). This requires operation under vacuum, as
air would scatter the electrons. At the top of the microscope column is an electron
gun and a system of electromagnetic lenses focuses the electron beam on the sample
[2]. Typical instruments are capable of voltages from 40 to 120 kV and micro-
scopes in the range of 200400 kV are becoming more common. A high resolution
is possible because of the short wavelength of the electrons. The resolution in TEM
is rather limited by the properties of the specimens than by those of instrumentation.
Its present level is at 0.31.0 nm. The image contrast in TEM is obtained due to
electron scattering. The absorption of electrons into the specimen is unusual, but
electrons scattered to large angles do not contribute to the image in the usual bright
field mode and thus appear to be absorbed. In the case of ordered or crystalline
sample material, this results in diffraction contrast, which is strongly dependent on
the crystal orientation. In amorphous materials, a mass thickness contrast is formed,
where the image brightness depends on the local mass thickness. Thus, darker
regions in the bright field image mode are regions of higher scattering.
1.2.1 Analytical TEM
The technique of electron energy-loss spectroscopy (EELS; Fig. 1.2) is of great

interest for soft matter analysis, especially in combination with cryo-preparation
techniques (Cryo EELS). Cryo EELS represents a valuable approach to analyse
both the morphology of a sample and its chemical composition since the energy
spectrum of electrons passing through the specimens also contains information
about the element composition. When the electron beam impacts the sample atom,
some of the electrons are inelastically scattered and lose a part of their energy.
Every element possesses its own specific energy loss. Thus, the elemental com-
position of the particle or droplet can be determined by analysis of this specific
energy by means of a spectroscope attached to the electron microscope [3]. Based
Fig. 1.2 Schematic representation of the basic principles of a EELS and b EDX TEM techniques
on this phenomenon, EELS can be used for chemical analysis of various liquid
dispersions, including systems containing polymers.
Another technique which is widely used for the elemental analysis in combi-
nation with electron microscopy is energy dispersive X-ray spectroscopy (EDX).
When a high-energy electron beam impinges upon a specimen, X-ray photons are
produced. Characteristic X-rays have well defined energies which are characteristic
of the atoms in the specimen. Thus, the elemental composition of the investigated
sample can be determined. In most cases EELS is expected to offer higher spatial
resolution than EDX because the effect of beam broadening and aberrations of the
probe-forming lens can be controlled by means of an angle-limiting collection
aperture. The technique of EELS as well as energy-filtered transmission electron
microscopy (EFTEM) imaging are usually applied to the detection of light elements
such as C, N or O, where EDX is less sensitive. Isaacson and Johnson [3] provided
the theoretical basis for the prediction of detection limits in EELS by introducing
the concepts of minimum detectable mass (MDM) and minimum detectable mass
fraction (MMF). The MDM describes the smallest amount of material that can be
detected in a given matrix. A small beam diameter is desirable, thus the use of a
scanning transmission electron microscope equipped with a field emission source is
preferable [4]. Alternatively, the MFF represents the smallest concentration of
elements that can be measured in a given matrix. This parameter depends primarily
on the total current available in the probe. Thermal emission tips that provide large
beam currents and conventional transmission electron microscopy mode may
therefore be preferable. In the ideal case of a sample that is not susceptible to beam
damage, traces of chemical elements as low as 0.030.1 % can be detected when
using a 200 kV instrument. However, the cryo EELS detection limit is much lower,
thus rendering the analysis of colloidal compositions a challenging task. Frozen
hydrated systems like nanoemulsions or polymer solutions may contain only low
amounts of specific atoms like N, P, Cl or F. Thus, the statistical noise obscures the
weak energy-loss near edge structure (ELNES) signals leading to increased errors
in background extrapolation and further deterioration of the detection limits.
Moreover, cryo EELS usually requires the use of primary energies of 200 kV (in
contrast to the conventionally used 80120 kV for cryo TEM imaging) and high
1.2 Transmission Electron Microscopy 7
beam currents, which lead to complete beam damage of the specimen structure
before the adequate spectra can be recorded. Currently there are two general types
of energy filters [post-column (GIF) and column-integrated (omega)] which are
used for EELS/EFTEM analysis.
1.3 Scanning Electron Microscopy (SEM)
In SEM [57], the image is formed step by step by scanning a focused electron
beam across the specimen. The primary electrons penetrate the solid specimen and
are deflected by a large number of elastic scattering processes (Fig. 1.1). The
energy spectrums of electrons that leave the specimen are collected by the detector
system to generate specific information and types of contrast as detailed below:
The surface topography of the sample is primarily registered by secondary
electrons (SE), i.e., all emitted electrons with exit energies below 50 eV. Sec-
ondary electrons can emerge from the specimen only from within a thin surface
layer of a few nanometers. The image contrast depends on the selected angular
range of the electrons collected.
Material contrast can be obtained by back-scattered electrons (BSE, Fig. 1.1)
which possess energies between 50 eV and the primary energy at the point when
they pass through the surface of the specimen. This contrast results in an increase
in intensity with increasing mean atomic number [8].
The SEM technique has a few important characteristics which make it a highly
popular microscopic technique for the ultrastructural investigation of different
kinds of hydrated materials, including nanoemulsions. Firstly, 3D like images of
the sample surface can be obtained in this fashion, as opposed to the 2D projec-
tions of the section volume which can be obtained by TEM. Secondly, a great
depth of focus is achieved by SEM. At low magnifications, it can be in the range of
a few millimeters which is especially important for samples with high surface
corrugation. However, there are certain limitations. These include a lack of
internal details, a somewhat limited resolution and the risk of electron beam
damage. As for TEM, a certain protection of the sample in its natural state can be
obtained by cryogenic techniques of sample preparation (cryo SEM). The involved
procedures are analogous to those employed for sample preparation for cryo TEM
and are detailed in next chapters.
The benefits of cryo SEM include usefulness for the analysis of certain types of
colloidal systems which are not entirely suitable for cryo TEM investigation.
1.4 Atomic Force Microscopy
In an AFM [9], a constant force is maintained between the probe and sample while
the probe is raster scanned across the surface. By monitoring the motion of the
Fig. 1.3 Piezoelectric quartz tuning fork with attached sharp probe can both vibrate and detect
vibration changes with sensitivity enough to achieve atomic AFM resolution without external
mechanisms for vibration and optical (laser-photodiode) detection
probe as it is scanned across the surface, a topographical image of the surface is

constructed. Currently there are two main detecting mechanisms, which are used in
AFM. First one uses optical detection system. The optical lever operates by
reflecting a laser beam off the cantilever. Because the cantilever-to-detector dis-
tance generally measures thousands of times the length of the cantilever, the
optical lever greatly magnifies motions of the tip. A compensation network
monitors the cantilever deflection and keeps it constant by adjusting the height of
the sample (or cantilever) [10]. The second detection system is based on the usage
a quartz tuning fork (Fig. 1.3). Piezoelectric quartz tuning fork with attached sharp
probe can both vibrate and detect vibration changes with sensitivity enough to
achieve atomic AFM resolution [11] without external mechanisms for vibration
and optical (laser-photodiode) detection.
Usually AFM can be operated in two modes. First one is a contact mode.
Contact mode AFM is mainly used to image hard surfaces when the presence of
lateral forces is not expected to modify the morphological features. These
attractive forces include capillary and adhesive forces, as well as Van der Waals
forces. They result in part from a thin layer of molecules adsorbed on the sample
and the tip. The second mode of conventional AFM is tapping mode. In tapping
mode, the cantilever is driven to oscillate up and down at near its resonance
1.4 Atomic Force Microscopy 9
frequency by a small piezoelectric element mounted in the AFM tip holder. The
amplitude of this oscillation is typically 100 to 200 nm. Due to the interaction of
forces acting on the cantilever when the tip comes close to the surface, Van der
Waals forces or dipoledipole interactions or electrostatic forces etc. cause the
amplitude of this oscillation to decrease as the tip gets closer to the sample.
A Tapping AFM image is therefore produced by imaging the force of the oscil-
lating contacts of the tip with the sample surface. AFM can also image the softness
of a sample by pressing the cantilever into it at each point in a scan. The scanner
raises the sample or lowers the cantilever by a pre-set amount, the modulation
amplitude (usually 110 nm). In response, the cantilever deflects an amount
dependent on the softness of the sample: the harder the sample, the more the
cantilever deflects. If instead of amplitude change one detects a phase shift, such
method is named phase imaging. It is necessary to mention here that there are a
large number of more specific mechanisms of image contrast formation, like
electrostatic, magnetic, Kelvin force etc. The inquisitive reader may find a detailed
description of those techniques in literature [10].
References
1. Ruska, E., Knoll, M.: Das Elektronenmikroskop. Z. Phys. 78, 318339 (1932)
2. Sawyer, L.C., Grubb, D.T.: Polymer Microscopy, 2nd edn. Alden press, Oxford (1996)
3. Egerton, R.F.: Electron Energy-Loss Spectroscopy in the Electron Microscope. Plenum Press,
New York (1986)
4. Isaacson, M., Johnson, D.: Ultramicroscopy 1, 3352 (1975)
5. Ardenne, M.: Z. Phys. 109, 553572 (1938)
6. Ardenne, M.: Z. Tech. Phys. 19, 407416 (1938)
7. Knoll, M.: Z. Tech. Phys. 16, 467475 (1935)
8. Reimer, L.: Image Formation in Low-Voltage Scanning Electron Microscopy. SPIE optical
engineering press (ed by D. OShea), TT12
9. Binnig, G., Quate, C.F., Gerber, C.: Phys. Rev. Lett. 56, 930 (1986)
10. Wiesendanger, R.: Scanning Probe Microscopy and Spectroscopy. Cambridge university
press, Cambridge (2003)
11. Giessibl, F.G.: Science 267, 6872 (1995)
Part II
Biological Related (Hydrated) Matter
Chapter 2
Visualization of OrganicInorganic
Nanostructures in Liquid
2.1 Introduction
The characterization of colloidal systems like pharmaceutical or hydrated

chemical formulations by microscopic techniques is essential to obtain reliable
data about the actual morphology of the system. Since the size range of col-
loidal drug delivery systems has long ago reached the lower end of the
nanometer scale, classical light microscopy has been replaced by electron
microscopy techniques which provide sufficient resolution for the visualisation
of nano-sized structures. Indeed, the superior resolution and methodological
versatility of electron microscopy has rendered this technique an indispensable
tool for the analysis of nanoemulsions. Microscopic analysis of these lipid-
based drug delivery systems with particle sizes in the lower submicron range
provides critical information about the size, shape and internal structure of the
emulsion droplets. Moreover, surfactant aggregates such as liposomes or mul-
tilamellar structures which remain unnoticed during particle size measurements
can be detected in this fashion. This chapter provides a brief overview about
both transmission electron microscopy (TEM) and scanning electron micros-
copy (SEM) techniques which have been employed to characterise colloidal
solutions. Of special interest are sophisticated cryo techniques of sample
preparation for both TEM and SEM which deliver high-quality images of
pharmaceutical formulations in their natural state. An overview about the
instrumentation and sample preparation for all presented methods is given.
Important practical aspects, sources of error and common artefacts as well as
recent methodological advances are discussed. Selected examples of electron
microscopic studies of nanoemulsions are presented to illustrate the potential of
this technique to reveal detailed and specific information.
The colloidal solution structure can be viewed in an electron microscope without
further preparation and certain conclusions can be drawn from the obtained images
if the circumstances are favourable. However, far more representative information

14 2 Visualization of OrganicInorganic Nanostructures in Liquid
Fig. 2.1 Cryo TEM of different hydrated systems. a nanoemulsion, b liposomes-proteins

solution, c solid lipid nanocaring system, d liquid crystal structure (Scale bar 20 nm), e self-
assembled oil in water system functionalised with silica particles (Scale bar 50 nm)
can be gained upon observation of the sample in its native state after cryo-prepa-
ration in thin vitrified layers or at a fracture plane using both transmission electron
microscopy (TEM) and scanning electron microscopy (SEM) (Fig. 2.1) [1].
Although some of the described techniques are rarely employed for nanoemulsion
characterisation due to the time-consuming and complex sample preparation and
high costs, all methods reported in the literature are briefly described here. Since
TEM in combination with cryogenic techniques of sample preparation is among the
most suitable methods for the investigation of nanoemulsions in their original state,
a focus is laid on these and related techniques. The innovative techniques of
electron energy-loss spectroscopy (EELS) and energy dispersive X-ray spectros-
copy (EDX) are likewise presented in this context. Other microscopic techniques
such as atomic force microscopy (AFM) have been employed for the investigation
of colloidal solution. However, the output of this technique is more essential for the
investigation of high pressure frozen and freeze substituted biological systems than
for colloidal systems (see Chap 4, 5, 6, and 7). The electron microscopic techniques
that are most frequently employed for the analysis of nanoemulsions, nanosus-
pensions, polymer solutions etc. today as well as recent methodological advances
are elucidated in the following sections.
2.2 Analytical Techniques Employed for the Characterisation of Colloidal Systems 15
2.2 Analytical Techniques Employed for the Characterisation

of Colloidal Systems
The characterisation of nano-sized colloidal systems requires diverse techniques

and a certain experience. Basic formulation characteristics such as visual
appearance, pH, mean particle size, particle surface charge, chemical stability of
employed excipients and the localisation of incorporated drugs provide useful
information [2]. Especially, the mean particle size and the particle size distribution
are frequently employed to characterise the long-term stability of novel formula-
tions. These parameters can be determined by optical light scattering techniques.
More specifically, dynamic light scattering (DLS, photon correlation spectros-
copy) is frequently employed for determination of nano-sized oil droplets within
emulsions. Among the obtained results, the mean particle size as intensity-
weighted mean of the hydrodynamic diameter and the polydispersity index (PDI)
are mostly presented [3, 4]. The latter characterises the width of the particle size
distribution and thus the homogeneity of the formulation. A small PDI below 0.2
indicates a narrow droplet size distribution and thus better stability against de-
stabilisation phenomena such as Ostwald ripening [2]. The characterisation and
stability assessment of nanoemulsions is strongly associated with their droplet size
and PDI. If both parameters remain largely unchanged during a prolonged
observation period, a formulation is usually considered physically stable [5].
2.3 Transmission Electron Microscopy: Experimental Setup,

Sample Preparation and Potential Artefacts
For hydrated samples such as nanoemulsions, the factors affecting the preservation
of the structural integrity are identical in both TEM and SEM [6]. Shrinkage of the
colloidal system due to complete dehydration and drying usually causes strong
structural changes. As a result, the final electron microscopic images may describe
completely modified structures which have nothing in common with the original
formulation morphology. Techniques based on cryofixation and low temperature
electron microscopy help to overcome these major problems.
If cryo TEM is not available, a conventional negative staining analysis with or
without dilution can be performed on nanoemulsions. Staining techniques are
frequently employed for imaging with TEM since they are easy, fast and uni-
versally applicable. The most common staining agents are salts of heavy metals
such as molybdenum, tungsten or uranium which possess atomic numbers between
42 and 92. These agents must be benign to the wet specimens, form a thin glassy
layer upon drying and must resist electron beam radiation damage to a satisfying
extent. During sample preparation, a droplet of the nanoemulsions is placed on a
carbon coated grid onto which it is rapidly adsorbed. Subsequently, an aqueous
solution of a heavy metal salt is applied for staining. The sample is then left to dry
and finally observed by TEM at room temperature. This technique allows for the
identification of the dehydrated shells of the nanoemulsion droplets which are
stabilized by surfactant. The strongly scattering metal ions form an amorphous
shield enveloping the weakly scattering structural features to enhance the electron
microscopy contrast. A high reverse contrast is thus seen in bright field TEM
images, with light droplets against a darker background. Negative staining can be
used to visualise the size, shape and internal structure of the sample. The negative
stain not only provides contrast for weakly scattering specimens, but also physical
support against collapse of the sample structure during drying and protection
against electron beam damage. Such sample can be also use for the topographical
EFTEM characterisation (see below).
However, it should be kept in mind that conventional electron microscopy is prone
to artefacts in case of surfactant solutions, i.e., hydrated colloidal dispersions. Both
drying and staining techniques can affect the structure and morphology of the sample;
thus, great care should be taken during interpretation of the obtained images. Con-
ventional negative staining on continuous carbon support films bears the risk of
sample distortion due to adsorption and flattening during the drying of the thin
aqueous film of negative stain or evaporation in the TEM. Apart from adsorption
artefacts, variable spreading and incomplete specimen coverage by the stain solution
can lead to non-uniform staining results. Specimen distortion from surface tension
forces during evaporative drying or the formation of a saturated salt solution before
the final drying may occur as well. Ice crystal formation may occur during investi-
gation of the sample, which may lead to misinterpretations. Even the modified
technique of cryo negative staining bears the risk of selective particle orientation due
to interfacial forces and flattening of fragile structures despite the maintained sample
hydration and protection against electron beam damage. However, not all TEM
laboratories are equipped with cryo electron microscopy facilities. Therefore, the
application of air-dried negative staining techniques for biological specimens or
other aqueous systems such as colloidal dispersions remains justified.
2.4 Cryogenic Transmission Electron Microscopy (Cryo

TEM): Experimental Setup, Sample Preparation
and Potential Artefacts
Generally speaking, the electron microscopic technique of choice for an artefact-free

visualisation of nanodispersions in water is cryo TEM. Figure 2.1 represents a few
examples of the effective usage of cryo TEM for the detailed investigations of
nanoemulsions, liposome containing systems, liquid crystals, self assembled struc-
tures functionalised with silica particle etc. By means of a complex sample prepa-
ration, the formulation microstructure is displayed in its original state and a clear
differentiation between nano-sized oil droplets and other structures can be obtained.
An equally suitable alternative is freeze-fracture TEM which will be discussed later.
2.4 Cryogenic Transmission Electron Microscopy (Cryo TEM) 17
Although water is the most abundant component of biological material, it is

inevitably excluded from conventional electron microscopy since it evaporates
rapidly under the vacuum conditions of an electron microscope. The development
of cryo-electron microscopy of vitrified specimens has radically changed the sit-
uation over the last decades [7, 8]. The discovery of vitrification opened the door to
a number of further developments which greatly facilitated cryo-electron micros-
copy studies. Simple methods were found for preparing a thin vitrified layer of
aqueous specimens [9, 10]. The cutting of vitrified sections, thin enough for high
resolution observation, was also found possible. Among the various kinds of cryo
specimen holders and plunge freezing devices, those which were of a simple nature
and easy to handle turned out to give the best results. It was also observed that an
optimal use of phase contrast could compensate to a large extent for the inherently
low contrast of unstained vitrified specimens. Furthermore, a rapid drop in tem-
perature during freezing provides the possibility to capture hydrated specimens in
their momentary movement without changing their structure and helps to reduce the
effect of electron beam damage. One of the basic requirements for TEM is that the
specimen be very thin. This can be achieved by the technique of cryo-sectioning
using high pressure freezing devices after the bulk sample has been vitrified [11,
12]. Alternatively, liquid suspensions can be prepared in the form of a thin film
which is subsequently vitrified as a thin layer. The first method can generally be
applied for various tasks, but is rather complex and demanding in nature. The
second method is simple and rapid, but is limited to the analysis of liquid sus-
pensions of moderate viscosity with particle diameters of less than 200 nm.
The sample preparation for cryo TEM involves three main steps: A small
aliquot (approximately 3 ll) of a fluid suspension containing the sample is applied
to the surface of a supporting substrate such as a holey or continuous carbon film
that is attached to the surface of a standard TEM specimen grid. Subsequently, the
droplet is carefully blotted with filter paper until most of the supernatant liquid is
removed and only a thin layer of approximately 100 nm thickness is left on the
support substrate. On perforated carbon support films, the thin sample film is left
stretched over the holes.
The thin fluid layer is rapidly immersed into a suitable cryogen of high heat
capacity, which leads to instantaneous and contaminant-free freezing. This vitri-
fication or shock-freezing by propelling the grids into a cryogen is ideally per-
formed by means of a plunging device and a humidity- and temperature-controlled
environmental vitrification system. The vitrified samples then need to be trans-
ferred into the TEM cryo holder of the microscope under liquid nitrogen and are
examined at temperatures around 100 K.
There are two key factors regarding specimen preparation that are critical to
obtaining high quality cryo TEM data, assuming of course that biochemical
integrity of the specimen is given: the proper preparation of the support substrate
and the considerate blotting of the sample droplet to a thin fluid layer on the
substrate prior to freezing. Once the thin film containing the specimen is produced,
it is immediately plunged into a suitable cryogen. A cryogen that is commonly
employed for this task is liquid ethane. Liquid nitrogen is used to maintain the
temperature of the ethane near its melting point of -183 C. With a freezing rate
around 1,000,000 K/sec, the fluid surrounding the specimen does not have time to
form crystalline ice, which would damage the fragile sample; it is vitrified instead
[8]. Embedded within this layer of vitreous ice, the specimen is essentially pre-
served in its native state at near atomic resolution.
However, there are certain limitations to the applicability of this technique.
Artifacts may emerge during the freezing process, such as ice crystal formation or
modifications due to humidity or temperature changes. For an excellent overview
about the variety of potential artifacts in cryo TEM, the reader is referred to the
recent review by Kuntsche et al. In addition, the maximum specimen thickness that
can be observed is limited to a few hundred nanometers. Thus, cryo electron
micrographs of polydisperse systems may be biased towards small particles due to
the preparation technique. The specimen preparation involves application of the
liquid sample on the microscopic grid and removal of the surplus liquid with filter
paper until an ultra-thin sample film remains in the holes of the grid, particularly in
their centre. Structures which exceed the thickness of this film are either removed
or relocated to thicker areas of the film during this procedure. Unfortunately, areas
of increased thickness are often too sensitive towards the electron beam to deliver
reliable results upon investigation. As a consequence, aggregates or droplets with
large dimensions may remain undetected. Thus, a direct comparison of droplet
sizes as observed in cryo TEM with the results of particle size measurements by
DLS or laser diffraction should be performed with great caution. The data obtained
by cryo TEM should be regarded as complementary qualitative information about
the shape and size of the observed particles. A quantitative evaluation of cryo
electron micrographs of a certain formulation aiming at an accurate size distri-
bution of the observed droplets would require evaluation of large amounts of
individual images and specific programmes.
Apart from these projection issues, the contrast of electron microscopy is
comparatively poor and may require additional staining techniques. Despite a
certain protection due to the cryo-fixation of the samples, severe electron beam
damage of frozen materials may occur as well, resulting in bubbling of the
sample and out-of-focus images.
Nevertheless, cryo TEM of frozen-hydrated unstained specimens is presently
among the preferred approaches for high-resolution studies because it provides
data on the fully native structure and, as indicated, some protection of the speci-
mens against electron radiation damage. Cryo TEM is particularly useful to
investigate structural details of colloidal nano-sized systems, e.g., to detect the
presence of vesicles among nanoemulsion droplets (Fig. 2.1). The fine ultra-
structure of both ordered and non-ordered multilamellar structures can be resolved
(Fig. 2.2). A lot of questions concerning the membrane organisation of the lipid-
drug containing systems also can successfully addressed by cryo TEM study
(Fig. 2.3). Overall, oil droplets are comparatively simple to be distinguished in
cryo TEM images. They always appear as spherical dark droplets while solid lipid
nanoparticles, liposomes or other related lipid structures may appear as needle- or
rod-like structures when viewed edge-on. The systems can be investigated with or
2.4 Cryogenic Transmission Electron Microscopy (Cryo TEM) 19
Fig. 2.2 Self assembled oil in water system (lamellar phase): cryo TEM images and TEM
intensity profiles
without dilution. In general, dilution of the investigated nanoemulsions is advis-

able since examination of undiluted samples frequently leads to crowded images
where individual structures cannot be clearly characterised.
Cryo TEM study in correlation with conventional negative staining can bring
up new aspects of sample characterisation. For example, astonishingly interesting
results could be obtained for conventional nano-sized oil-in-water emulsions by
investigation with TEM at room temperature after negative staining with uranyl
acetate (Fig. 2.4). A direct comparison with images of the same samples obtained
by cryo TEM suggests that the morphology of the systems is comparatively well
preserved despite the unfavourable surroundings within the TEM. Summarising
our experiences with TEM analysis of nanoemulsions at room temperature, it may
be assumed that it is not primarily the nature of the oil which is decisive for the
quality of the obtained images, but the efficacy of the employed surfactant to
stabilise the oil droplets. In Fig. 2.4, a conventional cosmetic oil of a molecular
weight around 300 g/mol was emulsified using different amounts of a sucrose ester
surfactant. Perfectly clear phase boundaries of the droplet shells after evaporation
in the TEM were found for systems of an ideal surfactant concentration of
2.5 % w/w and high physical stability. A surplus of surfactant did not improve the
formulations general properties, but rather destabilised the system by introducing
aggregates and leading to droplet deformation. The obtained images of both
Fig. 2.3 Cryo TEM images of liposomes including membrane incorporated drug molecules
formulations corresponded very well to the native structures as observed by cryo

TEM. It may thus be concluded that TEM images obtained at room temperature
provide reliable information about the quality of the investigated nanoemulsion,
i.e., the stability of the emulsified oil droplets, even though an exact visualisation
of the droplet shape remains confined to cryo TEM.
2.5 Laser Light Scattering Versus Electron Microscopy
DLS exhibits certain limitations and may provide incomplete information. Firstly,
it may fail to recognise the presence of a small population of large droplets present
in nanoemulsions. Likewise, other surfactant aggregates (Fig. 2.5) such as lipo-
somal (Fig. 2.6) vesicles or lamellar structures are not detected; the exact com-
position of the colloidal system thus remains unknown. However, such structures
are frequent by-products of high-pressure homogenisation and should be
accounted for [4]. Moreover, the shape of the analysed oil droplets is usually
assumed to be a perfect sphere for calculation of the DLS results, which is not
always the case. Thus, determined particle sizes for droplets of variable shape may
not be entirely representative. Furthermore, most samples have to be diluted prior
to DLS measurements to ensure sufficient transparency for accurate droplet size
determination. As a consequence, reversible destabilisation phenomena such as
flocculation or the appearance of larger aggregates may remain unnoticed. In order
to account for these issues, additional techniques of analysis are highly recom-
mendable. Sophisticated methods such as sedimentation, field flow fractionation,
nuclear magnetic resonance spectroscopy or Fourier transform infrared spectros-
copy have been proposed in this context. However, the microscopic visualisation
2.5 Laser Light Scattering Versus Electron Microscopy 21
Fig. 2.4 Comparison of cryo TEM and conventional TEM after negative staining with uranyl
acetate: nanoemulsions stabilised by either 5 % (a and b) or 2.5 % (w/w) of sucrose stearate
(c and d) were investigated with both methods. On the left hand side (a, c), the cryo TEM images
are given. On the right hand side (b, d), the corresponding images obtained by conventional TEM
at room temperature are given
of the investigated nanoemulsions might represent the most reliable and infor-
mative method for formulation characterisation.
When employing microscopic techniques for nanoemulsion characterisation,
the presence of larger droplets is not an entirely uncommon observation [13, 14],
albeit a rarely reported one. Experience has shown that it is possible to obtain
excellent DLS data for nanoemulsions over months of stability monitoring while a
microscopic analysis of the same sample reveals a definite change of the internal
structure. Recently, Preetz and co-workers [14] demonstrated the importance of
microscopic analysis for the characterisation of nanoemulsions and nanocapsules.
It was found that the mean droplet size determined by DLS was around 150 nm for
all investigated systems. In contrast, freeze-fracture TEM revealed variable droplet
Fig. 2.5 Cryo TEM image of self-assembled oil in water system (hexagonal phase). The oil
droplets are surrounded with attached surfactant bobbles
Fig. 2.6 Cryo TEM images of the liposomes-protein complex. a The area of the sample where
liposomes are not aggregating, b the area containing dense aggregates
sizes between 50 and 500 nm with the highest frequency around 100 nm, which
was additionally confirmed by atomic force microscopy.
Thus, the importance of microscopic techniques for the analysis of nano-
emulsion droplet size and overall morphology needs to be emphasized. Cryo TEM
is certainly among the most useful techniques for this task since it delivers detailed
information about the internal structure of the observed colloidal systems in their
native state. However, it is important to note that the studied images have to be
representative of the whole sample. Image analysis software should be employed
only for systems with a suitable contrast and composition. Several rounds of
2.5 Laser Light Scattering Versus Electron Microscopy 23
analysis are highly recommendable. A good overview of the investigated systems

can be obtained by combination of DLS or static laser diffraction and cryo TEM.
2.6 Freeze-Etching and Freeze-Fracturing of Nanoemulsions

for Transmission Electron Microscopy (FFTEM):
Experimental Setup, Sample Preparation and Potential
Artefacts
Freeze-etching, freeze-fracturing and cryo electron microscopy of frozen fluids are

complementary techniques mainly because the respectively obtained information
is based on different mechanisms [8].
Freeze-fracture electron microscopy techniques have emerged during the 1950
to 1960s and have been successfully employed for the analysis of hydrated
specimens for well over 30 years [15, 16]. The freeze-fracture technique consists
of physically fracturing a frozen biological sample. The structural detail exposed
by the fracture plane is visualised by vacuum-deposition of platinumcarbon to
make the replica for examination in a TEM.
The key steps involved in this procedure are rapid freezing of the sample,
fracturing, replication and replica cleaning. The rapid freezing, i.e., cryofixation,
of the nano-sized suspension is usually performed by swiftly immersing the
sample into a liquid coolant such as subcooled liquid nitrogen. In this context, pre-
treatment with cryoprotectants such as glycerol is sometimes necessary to avoid
ice crystal damage. Chemical fixation with glutaraldehyde beforehand serves to
avoid artefacts induced by the cryoprotectant. In many cases, successful freezing
of hydrated samples requires ultrarapid freezing techniques, such as optimised
plunge freezing, jet freezing, spray freezing, high-pressure freezing or freezing by
impact against a cold metal block [16]. Subsequently, the fracturing of the sample
is carried out under vacuum at liquid nitrogen temperature by breaking the sample
in a hinged device or by using a liquid nitrogen-cooled microtome blade. If
deemed necessary, an additional etching step may be performed which consists of
vacuum sublimation of ice after fracturing. In other words, the ice can be removed
from the surface of the fractured specimen by freeze-drying by increasing the
temperature to about -100 C for several minutes to let ice sublime.
The replicas are then prepared by shadowing and backing of the specimen. The
surface of the sample is usually shadowed with platinum to achieve a good
topographic contrast and then covered with a strengthening layer of electron-lucent
carbon to stabilise the ultra-thin metal film. More specifically, the cold fractured
surface, possibly etched, is shadowed with evaporated platinum or gold at an
average angle of 45 in a high vacuum evaporator. A second coat of carbon,
evaporated perpendicular to the average surface plane, is often performed to
improve stability of the replica coating. The topographical features of the frozen,
Fig. 2.7 Freeze-fracture TEM micrographs of different lecithin-based nanoemulsions. Images

reprinted from [19] with kind permission from Springer Science and Business Media. The
influence of increasing contents of glycerol within the formulation is demonstrated by decreasing
particle sizes and a more homogeneous droplet size distribution. Coalescence phenomena can be
detected in image a
fractured surface are thus transformed into variations in the thickness of the
deposited platinum layer of the replica.
The specimen is then returned to ambient temperature and pressure and the
extremely fragile pre-shadowed metal replica of the fracture surface is released
from the underlying biological material by careful chemical digestion with acid
solutions or detergents. The still-floating replica is thoroughly washed free from
residual chemicals, carefully placed on fine grids, dried and then investigated in the
TEM. Further details and practical advice on freeze-fracture electron microscopy
can be found in the literature [16]. Overall, freeze-fracture electron microscopy can
be employed for the analysis of a large spectrum of different materials, including
liquids and dispersions, at intermediate to low resolution. Freeze-fracture TEM
(FFTEM) is well adapted to study lipid-containing colloidal suspensions, such as
liposomes, nanoemulsions and nanoparticles despite the relatively low signal to
noise ratio of the replicas. Polymer solutions, microemulsions and biological sys-
tems can be investigated as well. The most important feature of this technique is the
tendency of the fracture plane to follow a plane through the central hydrophobic
core of frozen membranes, thus splitting them in half. As a result, planar views of
the internal structure of the samples are obtained. As for all microscopic techniques,
care must be taken to avoid misinterpretation due to artefacts. Freeze-fracturing
techniques are complex in nature and the different steps of sample preparation, such
2.6 Freeze-Etching and Freeze-Fracturing of Nanoemulsions for Transmission 25
Fig. 2.8 Cryo SEM image of hydrated protein system. The sample has been plunge frozen in
liquid nitrogen, partially freeze dried, Pt coated and investigated at low temperature by high
resolution SEM
as chemical fixation, cryoprotective pre-treatment, cryofixation, freeze-fracturing,

etching and replication, may significantly influence the appearance of the investi-
gated sample. Therefore, considerate specimen preparation is essential to ensure
reproducible and reliable microscopic data.
The morphology of a lecithin-based nanoemulsion for topical application was
investigated by FFTEM following a standard protocol of sample cryofixation,
freeze-fracturing, freeze-etching and covering with platinum/carbon. The influence
of increasing amounts of glycerol within the systems was clearly shown (Fig. 2.7).
The droplet size, shape and size distribution could be monitored very well by this
technique. Likewise, instability phenomena such as the agglomeration of droplets
could be detected.
2.7 Scanning Electron Microscopy, Cryo SEM and Freeze-

Fracture SEM: Experimental Setup, Sample Preparation
and Potential Artefacts
Indeed, cryo SEM of freeze fracture-freeze dried samples is the best solution for
hydrated systems, which are highly viscous and/or have a strong tendency to
aggregation. Cryo SEM in combination with freeze drying will be also an only
solution when colloidal hydrated systems are used as a coating layer for
improving the adhesion of cells etc. Figure 2.8 represents a freeze dried cover
glass which was coated with dense protein layer. SEM micrographs clearly show
the protein layer morphology. On the one hand, an ultrathin layer of such a
Fig. 2.9 ATEM characterisation of the nano- a and macro b emulsions containing titanium
dioxide particles. c represents EFTEM 2D map of titanium (green) and oxygen (red) destitution,
d show a EDX spectra of nanoemulsion, and e low loss region of EELS spectra obtained from the
titanium dioxide aggregate [17]
solution for cryo TEM investigation can hardly be obtained. Viscous protein
network are adsorbed firmly onto the carbon coated grid and are not readily
removed by filter paper. The resulting layer may not be thin enough to be
transparent for the electron beam. Thus, cryo SEM provides a more adequate
impression of the overall morphology of inhomogeneous and/or viscous hydrated
biological and pharmaceutical systems.
2.8 Recent Advances: Cryo Analytical TEM (cryo ATEM) 27
Fig. 2.10 TEM and EFTEM of negatively stained nanoemulsion: a elastic filtered TEM image,
b EFTEM image, which was obtained at plasmon region (2030 eV), c relative thickness map
(t/k), d carbon K map, e CPR (carbon map/bulk Plasmon) image, f EFTEM uranium map (U)
2.8 Recent Advances: Cryo Analytical TEM (cryo ATEM)
It has to be mentioned that ATEM including EELS, EFTEM and EDXS has not yet
found broader application for pharmaceutical system characterisation. However,
EELS and cryo EELS can be extremely useful for nanoemulsions containing
additives such as pharmaceutical substances or functionalised mineral particles.
The localisation of the additives can be determined without damaging the native
structure of the system. Work is in progress in this area. Figure 2.9 elucidates the
importance of ATEM for the identification of metal particles within a nano- or
emulsion solution. Usage of EFTEM allows one to obtain a precise 2D map of the
titanium dioxide particle distribution. EDX spectra usually gives an impression
about all chemical elements which are present in the irradiated area of the sample
in a concentration higher than 0.20.5 w/w. EELS spectra from the low loss region
clearly shows a pronounced ELNES features of titanium dioxide. It has to be
mentioned that in most of the cases an accurate comparison between obtained and
reference spectra from the EELS database may give a clear impression about the
chemical state of a different component of the colloidal system. For example
silicon, which is very often used in pharmaceutical systems, can appear as a
hydrated silica or silicon oxide. Both forms have pronounced ELNES features
which can be easily identified using a reference spectra from EELS atlas incor-
porated in Digital Micrograph software [17].
The most astonishing usage of EFTEM can be assigned to the determination of

a topographical profile of negatively stained organic materials (Fig. 2.10) [18].
The correlative surface profile of polymer and biological materials and its volume
projection along with chemical composition with nanometer special resolution
have important implications for friction, lubrication, adhesion, macromolecular
interaction and any application involving (bio-) polymer surface modification by
way of coatings, temperature or chemical treatments etc. So far, such correlative
analysis was not achievable due to absence of a high resolution imaging technique
which could provide such correlative study. Here, an analytical TEM method is
presented which allows one to extract a surface profile of negatively stained
organic materials. The fine metal replica formed by negative staining can be
mathematically extracted by dividing the images acquired in the bulk plasmon
energy region (which contain information mostly about carbon and uranium for
stained organic materials) and on the carbon K edge which shows carbon content
solely. The resulting ratio map (CPR) shows a fine distribution of uranium atoms,
which replicate the sample surface topography with nanometer precision. The
proposed CPR method allows one to detect a complementary transmission,
chemical and topographical information of the same area of 101000 nm thick
samples. The proposed method is easy to perform, does not require an additional
instrumentation set up and has a great potential for comprehensive investigation of
any kind of organic materials.
References
1. Mason, T.G., Wilking, J.N., Meleson, K., Chang, C.B., Graves, S.M.: J. Phys.: Cond. Matter
18, 635 (2006)
2. Klang, V., Valenta, C.J.: J. Drug Del. Sci. Tech. 21, 55 (2011)
3. Hoeller, S., Sperger, A., Valenta, C.: Int. J. Pharm. 370, 181 (2009)
4. Klang, V., Matsko, N., Raupach, K., El-Hagin, N., Valenta, C.: Eur. J. Pharm. Biopharm.
79(1), 58 (2011)
5. Kuntsche, J., Horst, J.C., Bunjes, H.: Int. J. Pharm. 417(12), 120137 (2011)
6. Mueller, M.: Encyclopedia of human biology 2, 721730 (1991)
7. Bouchet-Marquis, C., Hoenger, A.: Micron 42, 152162 (2011)
8. Steinbrecht, R.A., Zierold, K.: Cryo techniques in biological electron microscopy. Springer-
Verlag, Berlin (1987)
9. Lepault, J., Booy, F.P., Dubochet, J.: J. Microsc. (Oxford) 129, 89102 (1983)
10. Dubochet, J., McDowall, A., Menge, B., Schmid, E.N., Lickfeld, K.G.: J. Bacteriol. 155,
381390 (1983)
11. Moor, H.: Theory and practice of high-pressure freezing. In: Steinbrecht, R.A., Zierold, K.
(eds.) Cryo-Techniques in Biological Electron Microscopy, pp. 175191. Springer-Verlag,
Berlin (1987)
12. Mueller, M., Moor, H.: Cryofixation of thick specimens by high pressure freezing. In:
Mueller, M., Becker, R.P., Boyde, A., Wolosewick, J.J. (eds.) The Science of Biological
Specimen Preparation. SEM, AMF OHare, Chicago, pp. 131138
13. Hatanaka, J., Chikamori, H., Sato, H., Uchida, S., Debari, K., Onoue, S., Yamada, S.: Int.
J. Pharm. 396, 188193 (2010)
References 29
14. Preetz, C., Hauser, A., Hause, G., Kramer, A., Mader, K.: Eur. J. Pharm. Sci. 39, 141151
(2010)
15. Severs, N.J.: Nat. Protoc. 2, 547576 (2007)
16. Severs, N., Robenek, H.: Methods Cell Biol. 88, 181204 (2008)
17. Klang, V., Valenta C., Matsko, N.: (To be published 2012)
18. Matsko, N., Letofsky-Papst, I., Albu, M., Mittal, V., Hofer, F.: (To be published 2012)
19. Zhou, H., Yue, Y., Liu, G., Li, Y., Zhang, J., Gong, Q., Yan, Z., Duan, M.: Preparation and
characterization of a Lecithin nanoemulsion as a topical delivery system. Nanoscale Res.
Lett. 5, 224230 (2010)
Chapter 3
Macromolecular Distributions
in Biological Organisms In Vivo
3.1 Introduction
At present, the description of biological ultrastructure is more closely related to the

living state and is important as a complementary study of dynamic events in living
cells by fluorescent light microscopy. However, the ultimate resolution of the
optical microscopy is limited by a few hundred nanometres, which is by far not
sufficient for the ultrastructural investigation at the level of individual macro-
molecule. Till now, TEM of ultrathin sections was the main technique used to
address the problem, although the low electron microscopy contrast of biological
samples, the necessity to use a two-dimensional projection of the sample volume
and the issue of beam damage of sample strictly limit the number of topics which
could be assigned to this method. Meanwhile, scanning probe microscopy has
developed into a multifunctional technique suitable for characterization of
topography, adhesion, mechanical, and other properties of an object in a broad
range of environmental conditions (vacuum, liquid, gas) on a scale from hundreds
of microns to nanometers [1, 2]. Furthermore, the applicability of atomic force
microscopy in life sciences had become obvious shortly after the invention of this
technique. Over the years, an evident progress in structural and functional
investigation of isolated biomolecules and their supermolecular assemblies under
liquid, single molecule force spectroscopy, imaging of surface dynamics of native
biological membrane has been demonstrated [36].
Nevertheless, until now numerous attempts to characterize by AFM the internal
ultrastructure of cells and tissues did not provide information fully comparable to
TEM data [710]. Two main reasons closely related to each other have contributed
to this effect. The first one is a lack of suitable sample preparation technique that
determines the amount and quality of cellular ultrastructural details detected by
AFM [11]. Difficulties with an adequate interpretation of the obtained AFM data
are the second one [12]. While the instrument characteristics (probe geometry and
planar interface, signal-to-noise ratio) allow one to access structural details on a

32 3 Macromolecular Distributions in Biological Organisms In Vivo
sub-nanometer scale [1318], an ultimate resolution of biomolecule or organelle

architecture is determined by its topographical and/or phase profile in a context of
the whole cell structure. Therefore, the preservation of the integrity of a bio-
macromolecule during cell fixation (chemical fixation and dehydration or cryo-
fixation followed by freeze-substitution), stabilization (type of resin, procedure of
embedding) and surface preparation (block-face or sections, etching technique)
becomes extremely important for the high resolution AFM.
Cryogenic sample preparation techniques based on high-pressure freezing
provide the best way to preserve the ultrastructure of biological objects. High
cooling rate (freezing process takes normally about 0.05 s) provides the cryofix-
ation in such a way that cell suspension or tissue culture are maintained in their
living-like state. Thus, such structural alterations of the cellular constituents like
conformational changes of proteins, their partial or complete hydrolysis due to the
contact with aggressive fixatives, dimensional changes due to the loss of mem-
brane semi-permeability that occurs in chemical fixation, can be avoided [19, 20].
Freeze substitution allows one to dissolve and replace the ice of the frozen material
by organic solvent containing fixative (-s) at low temperature. In order to achieve
an optimum preservation of a biological material, the choice of fixative is crucially
important [11]. The epoxy and anhydride groups, present in epoxy resin embed-
ding formulation can be used as powerful stabilising agents during freeze-sub-
stitution [21]. The epoxides stabilize protein without degradation effects, in
contrast to aggressive fixatives like OsO4 or glutaraldehyde [11].
In AFM, the best detection of small structural details can be achieved after
sectioning, when a maximum of embedding material from the vicinity of a
copolymerized biological object has been removed. Treatment with ethanol can
easily elutriate free polymer chains, which are not involved in the polymer net-
work and the excess of a crosslinker which did not react with epoxy chains,
thereby revealing topographical subtleties of the sample under investigation [12].
There are other effective etching techniques based on chemical and electron beam
etching method [22, 23].
The interpretation of new results in any subdivision of microscopy in life
sciences is currently based on our knowledge about cellular ultrastructure that has
been obtained by transmission electron microscopy of ultrathin section of bio-
logical material. In TEM, the ultrastructure is visualized by staining with heavy
metals salts [24, 25]. Thus, only the structures, which react with the staining
agents, and which can be reached by the staining agents are detected [2426].
Consequently, cell organelles, membranes, protein filaments, and nucleic acids are
clearly observed, but many proteins in the cytoplasm of the cell are practically
invisible [25, 27]. AFM, on the contrary, provides information about the cell
constituents that are distributed on the surface of the section or block face.
Therefore, due to the nature of the collected signal the protein content in the
cytoplasm can be detected by AFM (in case when they are preserved during the
sample preparation), but cannot be reliably interpreted due to lack of knowledge
about their shape and distribution in TEM. Obviously, atomic force microscopy
seems to be the unique method for the detection of a protein state (i.e., protein
3.1 Introduction 33
Fig. 3.1 TEM a, b and AFM phase c, d and corresponding topographical e, f images of the high-
pressure frozen and epoxy freeze-substituted adult nematodes C. elegans. The images
demonstrate the cross-section of the worm that was frozen at the live state a, c, e, and at the
state of necrosis b, d, f. Height variation: 030 nm in e, 024 nm in f; phase variation: 05 in c,
01 in d. N nucleus
molecule distribution and architecture) of the investigated biosample. Neverthe-

less, the adequate interpretation of the AFM data requires an accumulated image
database of each cytoplasmic protein molecule and their complexes within cell that
detected and analyzed simultaneously by AFM and other microscopy techniques
(TEM, cryo SEM, and immunocytochemical analysis).
In this chapter, the unique ability of atomic force microscopy to detect cyto-
plasmic protein state within a cell is demonstrates, which can be used for the
control of the quality of the sample preparation procedure in terms of cellular
protein preservation. The role of different cell constituents in the formation of the
AFM image is considered, and the choice of optimal AFM settings is discussed.
Using combined AFM and TEM data, the influence of different freeze-substitution
protocols on the quality of the protein preservation is analyzed. In order to
facilitate the interpretation of AFM images, we have developed a novel procedure
of dual AFM/TEM images preparation and analysis, when one particular organelle
or biomolecule can be cut into two parts, one being used for AFM and another one
for TEM. This approach leads to a breakthrough in AFM image interpretation, and
allows one to reveal new ultrastructural aspects of macromolecular arrangement in
a cytoplasm area, going far beyond the possibility offered by AFM or TEM alone,
provided the cellular proteins and other macromolecules are not damaged during
the sample preparation procedure.
3.2 AFM Tuning Parameters Used for the Imaging

of the Epoxy Embedded Biological Samples
The AFM of the embedded biosample detects both the topography of the block
face surface which appears due to the relaxation of the tension inside the
embedded biomolecules during or immediately after the sectioning process, and a
phase shift which can be attributed to the different density of the pure epoxy resin
and copolymerized cell constituents, surrounded by it [12]. Compared to the height
images, the phase images provide better contrast of fine morphological and non-
structural features due to their high sensitivity to the surface imperfections. In
addition, on surfaces with local variations of mechanical properties, the phase
changes are even more informative. As it is evident from the Fig. 3.1, the most of
information about macromolecule location within the cytosol of a dead cell
(Fig. 3.1f) is missing in the height image (Fig. 3.1d). When the preservation of a
protein macromolecule is good (Fig. 3.1c, e) like in case when the cells were
frozen in an alive state, the differences between the phase and the height images
are not so pronounced. The reason is the dense protein matrix of an alive cell,
which means that the protein macromolecules are located close to each other. This
almost excludes the possibility to find the area, where the macromolecular com-
plexes are surrounded with pure embedding material of very different elastic
modulus, as it happens in a dead or badly preserved cell. Still the phase images
3.2 AFM Tuning Parameters Used for the Imaging 35
provide the more complete information about the content and distribution of the
cell constituents.
It should be also noted that the knife marks on the block face surface, which
deteriorate the AFM height image, are almost invisible on the phase one (Fig. 3.1f,
d). This provides an additional reason favoring using the phase images. Another
important point to note is the presence of a water film on nearly all surfaces in ambient
air that makes interpretation of the phase image doubtful. Avoiding such phenomena
is a prime necessity especially in the present system as it leads to partial loss of phase
contrast. Therefore, all AFM images were collected in a hard-tapping mode [29].
In this measurement, probes with force constants of around 20 N/m were applied.
The phase curves obtained with a test sample (tobacco mosaic virus and hard
polystyrene particles, embedded in epoxy resin) at different A0 (free air probe
oscillation amplitude) show that at small amplitude, the phase behavior is different
from that at a high amplitude. This is most likely due to the liquid contamination layer
on the sample surface. Phase shift increases with a decrease of the Asp/Ao ratio and
saturates at Asp/Ao values below 0, 1 (here Asp is a sample contact amplitude). In the
experiments with test samples, the phase shift behavior was found to depend sub-
stantially on the polymer density, which is related to the elastic modulus. The force
interaction with harder materials leads to large positive frequency and phase shift
(white regions), as compared with the interaction with a soft material (dark region).
Both were measured with respect to the frequency and phase of the freely oscillating
cantilever. Therefore, such tuning parameters (Asp/Ao B 0, 1) were kept during all
AFM measurements of embedded biopolymers. It guarantees that the phase shift
images emphasize macromolecule structure with superior details, which is barely
seen in the height image (Fig. 3.1f, d).
3.3 Dependence of the AFM Phase and Topographical

Contrast on the Integrity of Cellular Protein Molecules
Which of the cell constituents bring the highest contribution to the formation of
topographical and phase AFM contrast of the embedded biological object? This
question is one of the most important for macromolecular atomic force micros-
copy. In mammalian cell, nearby 70 % of cell weight is taken by water, 22 %
macromolecules (18 % are proteins, nearly 2 % nucleic acids and nearly 2 % are
polysaccharides). The rest are phospholipids and other lipids (5 %), miscellaneous
small metabolites (3 %), and inorganic ions (1 %) [30]. Therefore, the topo-
graphical profile on AFM images could represent mainly individual macromole-
cules and their complexes. This assumption is supported by the appearance of the
cytoplasm after cell death when disassembly of the cellular biopolymers occurs.
Figure 3.1 represents the AFM phase and topographical images of pharyngeal cells
of C. elegans, which were frozen in alive state (Fig. 3.1c, e), and 3 days after the
death occurs (Fig. 3.1d, f). Figures 3.1a, b represent TEM micrographs of the same
Fig. 3.2 TEM thin section a and AFM phase b images of block face of the conventionally
chemically fixed adult nematodes C. elegans. Section was stained by uranyl acetate and lead
citrate. Phase variation: 07 in b
Fig. 3.3 AFM height image

of block face of the
conventionally chemically
fixed liver tissue. Insert
shows the height variation of
the sample surface (along the
black line)
samples. The topographical image of the dead cells shows the homogeneous tiny
grain matrix of the cytoplasm, in contrast to the alive cells, which demonstrate
some structural organization. Obviously, the most informative are phase images.
The grains, which look homogeneous in Fig. 3.1f appear to have different hardness
in Fig. 3.1d. The cytoplasm matrix appears to contain association of the parts,
which are disconnected with each other. The distribution of the small light regions
3.3 Dependence of the AFM Phase and Topographical Contrast on the Integrity 37
Fig. 3.4 TEM a, c, e and AFM b, d, f phase contrast images of the a, b Freeze-substitution was
performed in acetone containing 2 % OsO4. The substitution medium was replaced by pure
acetone after having kept the sample at 25 C during 2 h. c, d Freeze-substitution was performed
in acetone containing 2 % OsO4. The substitution medium was replaced by pure acetone at 0 C.
e, f Freeze-substitution was performed in acetone containing 20 % Epon/Araldite mixture. Phase
variation: 04 in b, 08 in d, 05 in f. Scale bars equal 500 nm. N nucleus, G Golgi complex,
mf pharyngeal muscle filaments, m mitochondria, pm pharyngeal membrane, cm cell membrane
in the phase image corresponds to ribosome agglomerates in the TEM image

(Fig. 3.1b). The area around white islands appears to have the same hardness like
free polymer surrounding the nematode. A pronounced difference in cytoplasm
content between the biomaterial, which was frozen at the live state and dead state
is evident. The discernible part of the cytoplasm filler (Fig. 3.1c) was removed and
cytosol of a dead cell (Fig. 3.1d) looks relatively empty. The most probable reason
for this phenomenon can be native proteolysis that is associated with non-physi-
ological form of cell death or necrosis [31]. The processes of clumping and ran-
dom degradation of deoxyribonucleic acid macromolecules during necrosis are
slow, which may allow one to still detect the nuclear envelope containing the
DNA. The extracellular matrix comprising a variety of versatile polysaccharides
and proteins [30] can be observed in both samples, but appears different. While in
an alive frozen tissue, it looks dense and consists of easily distinguishable grains,
in the necrotic tissue, it shows smooth homogeneous structure. Nevertheless, it can
be argued that the protein macromolecules bring the most pronounced contribution
in the appearance of the biological structure of embedded biosample that can be
detected by AFM. The other macromolecules like nucleic acids and polysaccha-
rides can be detected by AFM, but play a minor role in the formation of the AFM
image as their content within a cell is relatively low compared to the protein
content. It has to be mentioned here that the AFM study of conventionally
chemically fixed different biological species clearly demonstrated that the mac-
romolecular preservation of those samples look very similar to the necrotic tissue
(Figs. 3.2 and 3.3). Both samples demonstrate almost complete absence of the
detailed ultrastructure of the cytoplasm. On the other hand, the height variation of
the block face surface of those objects is in a range of 35 nm which is comparable
with the height variation of pure epoxy resin. The reason for this effect can be in
the usage of OsO4 during chemical fixation. The particular role of osmium
tetroxide in the proteolysis will be discussed in the following section.
3.4 Correlative AFM/TEM Analysis of the Protein

Preservation in the Samples, Prepared in Accordance
with Different Freeze-Substitution Protocols
Most of the cell constituents undergo some changes during the fixation and
dehydration process. Proteins, the main constituent of a biosample, are most
sensitive to different chemical fixatives and to the procedure of dehydration. The
detection of the state of the protein macromolecules inside the cell is of immense
importance as it allows one to estimate the quality of structural preservation. In
TEM, the protein integrity can be estimated only indirectly. So far good TEM
staining contrast is considered as an evidence of a good structural preservation of a
biosample, which is not really justified [11, 12]. Lack of knowledge about the state
of internal cell proteins often leads to wrong conclusions concerning the quality of
structural information obtained by TEM. In order to analyze whether the good
staining contrast in TEM is a mark of the good structural preservation in terms of
macromolecular preservation, the three selected freeze-substitution protocols were
applied for the high-pressure frozen C. elegans. Using 2 %OsO4/acetone for 2 h at
room temperature provides the best contrast of the membrane lipid bilayers, when
nuclear, mitochondrial, Golgi, ER membranes can be detected as two clearly
visible lines (Fig. 3.4a, b). Uranyl/glutaraldehyde/acetone, uranyl/OsO4/acetone or
uranyl/acetone freeze-substitution protocols that are often used cannot provide
such strong membrane visibility [3234]. The epoxy freeze-substitution protocol
when 20 % Araldite/Epon embedding medium/acetone is first used as a stabilizer
(as e.g., OsO4) and then as embedding medium, was selected as the best for protein
preservation [11] (Fig. 3.4e, f). On the other hand, this method does not provide
such a strong membrane contrast like after OsO4 treatment during 2 h at room
temperature. Figures 3.2c, d correspond to the sample where OsO4 was replaced
by pure acetone immediately after the temperature had reached 0 C. Such freeze-
substitution protocol is known as standard one [28] and allows one to reach the
compromise between the structural preservation and the TEM contrast (a partial
3.4 Correlative AFM/TEM Analysis of the Protein Preservation 39
Fig. 3.5 Corresponding a AFM phase (block face) and b TEM (ultrathin section) images of C.
elegans, high-pressure frozen and freeze-substituted in acetone containing 20 % Epon/Araldite
mixture. Phase variation: 04 in b
protein degradation occurs anyway when osmium tetroxide are used, but the
replacement of the fixative at 0 C minimizes such influence) [25].
TEM images exhibit better membrane contrast after post staining for the sample
exposed to OsO4 in acetone for a longer period of time (Fig. 3.4a, c). Corre-
sponding AFM image clearly indicates that cytoplasm and organelles after OsO4
exposure for 2 h (Fig. 3.4b) consists of small and almost homogeneous grains.
Most of the ultrastructural details are observed to be lost. AFM phase contrast
shows almost the same density of the matrix for the pharyngeal area, which is
densely packed with different well-known proteins and organelles (actin and
myosin filaments, ribosomes etc.) and the intercellular space, which contains very
little proteins. Also, there is no difference between the grain density inside the
mitochondrial matrix, where the density of the protein packing is extremely high,
and outside of it.
AFM image of the sample, which was exposed in OsO4 for a shorter period of
time (Fig. 3.4d) demonstrates denser mitochondrion matrices. In addition, the
structure of the grains becomes more differentiated. Almost no grains could be
observed in mitochondrion and nucleus membranes, which appear solid. AFM
phase image of the sample fixed with epoxy instead of OsO4 (Fig. 3.4f) provides
structural information similar to TEM. The cell organelles in both images are
easily identified. Organelle matrices (mitochondria, nucleus, and pharyngeal
muscles) appear to be very solid and dense. Each ribosome can be identified as an
individual organelle.
The above results clearly demonstrate that macromolecular content of the
samples fixed with osmium tetroxide are partially destroyed. The degree of
damage depends on the time of the OsO4 exposure. So far, the comparison of AFM
Fig. 3.6 TEM a-AFM phase b complementary couples of images of the nucleus of high-pressure
frozen and epoxy freeze-substituted C. elegans. Phase variation: 05 in b. The white arrow
indicates a nuclear membrane which is densely packed with proteins
Fig. 3.7 TEM a-AFM phase b complementary couples of images of the mitochondria of high-
pressure frozen and epoxy freeze-substituted C. elegans. (1, 2, 3) indicate ribosomes, which were
cut by the knife during sectioning, and appear as the nearly equal parts in each of the images.
Phase variation: 05 in b
and TEM images led to conclusion that high stainability of the sample correlates
with macromolecular density of the cellular matrix. This means, the less dense
matrix is detected by AFM, the higher staining contrast observed by TEM. This
assumption appears logical as the heavy metal salts have an ultimate size in
nanometer scale [25] and their penetration deep into the section depends on the
density of the sample. As it is evident from above, TEM images of the heavy metal
stained sections cannot provide direct information about the state of the cytoplasm
proteins. In contrast, AFM technique provides fast and unaffected test of the
macromolecular preservation of the investigated sample.
3.5 Identification of the Cell Constituents in AFM Phase Image 41
Fig. 3.8 TEM a-AFM phase b complementary couples of images of the endoplasmic reticulum
of high-pressure frozen and epoxy freeze-substituted C. elegans. Phase variation: 05 in b. ER
endoplasmic reticulum
3.5 Identification of the Cell Constituents in AFM Phase

Image Using AFM and TEM Complementary Couples
of Images
While the estimation of the protein state of a biosample from the AFM image can
be performed by the appearance of such specific organelles like mitochondria,
nucleus, and/or cell membranes, which can be easily distinguished in any of AFM
and TEM images, the identification of most of the others cell organelles in the
AFM micrographs is not so simple. Most of our knowledge about the form and
structure of organelles came from TEM of the ultrathin sections, and till now there
is no parallel data obtained by AFM. For example, the ribosomes, due to the high
stainability of RNA, have very specific view in the TEM images: they appear like
the intensive black dots about 20 nm in diameter, which makes them easy to
identify. In AFM, which shows only the topographical/phase contrast, the differ-
ence between the ribosomes and other proteins, which have almost the same size
and hardness, is obscure. A possibility to use TEM and AFM complementary
couple of images (see the inset of Figs. 3.5, 3.6, 3.7, 3.8, 3.9, 3.10) solves this
problem, making AFM image interpretation easy, and provides more complete
information about the ultrastructural details of a biosample (gap junctions, outer
mitochondrion membranes and cristae, ribosomes etc.). Here, the crucial point is
that one particular organelle or biomolecule can be cut into two parts, one part
being used for AFM and another one for TEM. For AFM, we used the block-face
of epoxy fixed and embedded nematodes (Fig. 3.5a), while the last ultrathin sec-
tion was collected, post stained with uranyl acetate and lead citrate, and then used
for TEM (Fig. 3.5b). This is illustrated in Figs. 3.6, 3.7, 3.8, 3.9 which show some
organelles with a larger magnification (Fig. 3.6 demonstrates nucleus (N), Fig. 3.7
show mitochondria (m), and Fig. 3.8 show endoplasmic reticulum (ER)).
Fig. 3.9 TEM b, d-AFM phase a, c of images of the hypodermal cell of the high-pressure frozen
and freeze-substituted nematode C. elegans. a, b, c Freeze-substitution was performed in acetone
containing an epoxy and d an 2 % of OsO4. c, d corresponding AFM and TEM images of
multivesicular body
The data about relative consistency and arrangement of the main organelles
obtained from the AFM and TEM images are supplementary. From TEM micro-
graphs one can identify cell organelles, whereas some additional structural features
(mainly protein arrangement) can be obtained from the AFM phase image (see
next chapter). For example, parts of particular ribosomes (marked by 13 in
Fig. 3.7) can be easily distinguished in both AFM and TEM images.
However the architecture of organelles detected by AFM depends on the quality
of the macromolecule preservation. Thus, using of the TEM and AFM comple-
mentary couple of images cannot guarantee the adequate interpretation of the
AFM data when the cellular proteins are partially or completely destroyed during
3.5 Identification of the Cell Constituents in AFM Phase Image 43
Fig. 3.10 TEM a, c, d and AFM phase b of images the area of the cells contact (gap junction) in
the hypodermal cell of the epoxy fixed adult C. elegans. a, b show a complementary couple of
AFMTEM. c The sample was frozen for the vitrified state and epoxy freeze-substituted; d the
sample was frozen almost for the vitrified state and freeze-substitution was performed in acetone
containing 2 % OsO4. The substitution medium was replaced by pure acetone after having kept
the sample at 25 C during 2 h. Phase variation: 05 in b. gj gap junction, ER endoplasmic
reticulum
the sample preparation procedure [12]. Furthermore, the preparation of the com-
plementary couple of images, which is in general not trivial, becomes extremely
complicated in case, when the AFM image does not contain the significant mac-
romolecular ultrastructure, which can be easily recognized by TEM. Here it should
be also noted that with a novel devise based on the combination of AFM with an
ultramicrotome, which has been recently developed [35], the preparation proce-
dure can be significantly simplified.
3.6 Interpretation of the TEM Images Obtained

from the Epoxy Fixed Sample Using Complementary
AFM Data
It seems to be clear that the reliable information about the organization of the cell
constituents can be obtained only when a biological structure is well preserved. It
means that cell organelles appearance should be investigated it terms of both their
location within the cell and their macromolecular structure. While the location of
Fig. 3.11 AFM phase a and cryo SEM b images of high pressure frozen biological objects.
a Sample was frozen almost for the vitrified state and epoxy freeze-substituted. b After freezing
sample was partially freeze dried, carbon-platinum rotary shadowed and observed by high
resolution SEM at low temperature (image courtesy from Martin Mller, Electron microscopy
laboratory I, ETH Zrich). Arrows indicate cytoplasm protein complexes
cell membranes as well as other organelles can be easily observed on the OsO4
treated samples, their protein content is normally lost. When the macromolecular
preservation is good, it provides a biosample with a very dense protein matrix and
heavy metal salts have limited access to the polar groups of the lipid bilayer and to
the other highly stained specific groups to render it visible in TEM. In addition,
relatively poor stainability of the protein macromolecules itself does not allow one
to properly detect them by TEM [11]. This problem is one of the most difficult to
solve. Using AFM as a complementary to TEM microscopic technique gives us a
chance to detect protein content of the epoxy fixed biosample and reveals new
ultrastructural aspects in addition to TEM data.
This suggestion is supported by the AFM/TEM investigation of the multivesicular
body in the hypodermal cell of the epoxy fixed C.elegans. The AFM image
(Fig. 3.9a, c) demonstrates that this structure is a cluster of nearby 50 nm vesicles,
which are encapsulated in a bounding dense membrane [36]. The TEM image
(Fig. 3.9b) shows a slightly grey background without any significant structures
inside. The membrane lipid bilayer becomes visible in TEM image (Fig. 3.9d) only
when the sample was prepared according to OsO4 fixation protocol. As has been
mentioned above, a biosample after the treatment with osmium tetroxide during 2 h
at room temperature loses most of its internal proteins, including the membrane
proteins. Therefore, heavy metal salts easily find access to the polar groups of the
lipids and residual proteins to make them visible by TEM. Thus, TEM images
obtained from the epoxy fixed sample or from the OsO4 fixed sample separately
cannot provide comprehensive information about the internal structure of such
organelle. In contrast, AFM image contains both aspects of a structural organization
of a biological material: arrangement of the organelle and its protein content.
3.6 Interpretation of the TEM Images Obtained from the Epoxy Fixed Sample 45
The same phenomena can be observed in AFM/TEM appearance of the gap

junctions in the epoxy fixed cells (Fig. 3.10). Gap junctions are constructed from
transmembrane proteins, which form structures called connexons. When the
connexons in the plasma membranes of two cells in contact are aligned, they form
a continuous aqueous channel, which connects the two cell interiors [29].
Figure 3.10a indicates that staining of the interacting plasma membranes is not
homogeneous. There are some spots on both of them, which are not stained. AFM
image indicates that these spots correspond to the clearly visible white grains
nearby of the same size on the phase image (Fig. 3.10b). So far it should be noted
that when the specimen is frozen in the vitrified state (that means that the protein
structure has not suffered at all from the growing of ice crystals) and epoxy freeze
substituted (Fig. 3.10c), the TEM appearance of the gap junction as a line of the
separate grey spots becomes even more pronounced. Explanation described above
leads to conclusion that unstained spots in the TEM image and corresponding to
them white grains in the AFM image could represent individual transmembrane
proteins channels. In the TEM image of osmium tetroxide fixed samples the gap
junction appears in traditional way as four black lines (Fig. 3.10d), which con-
firmed the assumption that the macromolecule content in such a sample is lost.
3.7 Difficulties in the AFM Image Interpretation
In certain cases, the interpretation of the AFM images becomes very complicated.
Such details like mitochondria, ER or nucleus (Fig. 3.6, 3.7, 3.8) can be easily
identified in the both images, but some small grains in AFM phase image do not have
an analog in TEM micrographs. In most of the cases, these look like a grey back-
ground in stained sections. As the pure plastic surrounding the nematodes does not
contain such grains in the AFM phase image, these substances, therefore, should be
some biological material. Probably most of them are protein molecules that are not
stained in TEM. This assumption is supported by the SEM data obtained from high-
pressure frozen and freeze-dried biological samples (Fig. 3.11) [37]. The cytoplasm
space and cell components of such samples consist of different kinds of grains,
which look similar to the structures detected by AFM. Also it should be taken into
account that most of the cytoplasm proteins do not have high affinity to staining
agents and thereby cannot be resolved as separate molecules by TEM of thin sec-
tions. Thus, for an adequate interpretation of such structures, another method is
required. One of the possibilities is receptor-ligand binding labeling.
3.8 Conclusion
Because of the nature of the collected signal, atomic force microscopy proves to be
a unique tool to examine the macromolecular (mainly protein) state of the
embedded biological sample directly. In contrast to TEM, simplicity of this testing
procedure allows one to use AFM routinely in order to check the macromolecular
preservation of each sample, before the further TEM investigation is done.
Phase images, in contrast to the height ones, provide the best visualization of
fine morphological features of the embedded biosample and, at the same time,
contain less microtomy artifacts (knife marks), which makes them more preferable
to use.
Using AFM technique, it was shown that the high TEM stainability of the
biological sample is a result of a low macromolecular density of a cellular matrix.
It might take place when cytoplasmic and membrane proteins have suffered from
sample preparation procedure or native proteolysis occurs. The best protein
preservation takes place when epoxy resin is first used as a stabilizer during freeze-
substitution procedure, and then as an embedding medium.
The possibility to use complementary AFM and TEM complementary couple of
images strongly facilitates AFM image interpretation and makes it more com-
prehensive. Furthermore, if the macromolecular content of the sample is not
damaged during sample preparation procedure, this technique allows one to access
new ultrastructural aspects of a macromolecular arrangement in a cytoplasm area,
beyond the possibilities provided by AFM or TEM alone.
References
1. Binnig, G., Quate, C.F., Gerber, C.: Phys. Rev. Lett. 56, 930 (1986)
2. Drake, B., Prater, C., Weisenhorn, A., Gould, S., Albrecht, T.R., Quate, C.F., Cannell, D.S.,
Hansma, H.G., Hansma, P.K.: Science 243, 1586 (1989)
3. Mller, D.J., Bldt, G., Engel, A.: J. Mol. Biol. 249, 239 (1995)
4. Mller, D.J., Schabert, F.A., Bldt, G., Engel, A.: Biophys. J. 68, 1681 (1995)
5. Mller, D.J., Engel, A., Amrein, M.: Biosens. Bioelect. 12, 867 (1997)
6. Oesterhelt, F., Oesterhelt, D., Pfeiffer, M., Engel, A., Gaub, H.E., Mller, D.J.: Science 288,
143 (2000)
7. Melling, M., Hochmeister, S., Blumer, R., Schilcher, K., Mostler, S., Behnam, M., Wilde, J.,
Karimian-Teherani, D.: Neuroimage 14(6), 1348 (2001)
8. Nag, K., Munro, J.G., Hearn, S.A., Rasmusson, J., Petersen, N.O., Possmayer, F.: J. Struct.
Biol. 126(1), 1 (1999)
9. Yamamoto, A., Tashiro, Y.: J. Histochem. Cytochem. 42(11), 1463 (1994)
10. MacDonald, D.E., Markovic, B., Allen, M., Somasundaran, P., Boskey, A.L.: J. Biomed.
Mater. Res. 41(1), 120 (1998)
11. Matsko, N., Mueller, M.: J. Struct. Biol. 152, 92 (2005)
13. Mou, J., Yang, J., Shao, Z.: J. Mol. Biol. 248, 507 (1995)
14. Walz, T., Tittmann, P., Fuchs, K. H., Mller, D.J., Smith, B.L., Agre, P., Gross, H., Engel, A.:
J. Mol. Biol. 264, 907 (1996)
15. Reviakine, I., Bergsma-Schutter, W., Brisson, A.: J. Struct. Biol. 121, 356 (1998)
16. Scheuring, S., Ringler, P., Borgina, M., Stahlberg, H., Mller, D.J., Agre, P., Engel, A.: Embo
J. 18, 4981 (1999)
17. Walters, D.A., Cleveland, J.P., Thomson, N.H., Hansma, P.K., Wendman, M.A., Gurley, G.,
Elings, V.: Rev. Sci. Instrum. 67, 3583 (1996)
18. Schmitt, L., Ludwig, M., Gaub, H.E., Tampe, R.: Biophys. J. 78, 3275 (2000)
References 47
19. Moor, H.: In: Steinbrecht, R.A., Zierold, K. (eds.) Cryo-techniques in Biological Electron
Microscopy, Springer, Berlin (1987)
20. Mueller, M., Moor, H.: The Science of Biological Specimen Preparation. In: Mueller, M.,
Becker, R.P., Boyde, A., Wolosewick, J.J. (eds.) Scanning Electron Microscopy inc., AMF
OHare (1984)
21. Causton, B.E.: The Science of Biological specimen preparation for microscopy and
microanalysis. In: Mueller, M., Becker, R.P., Boyde, A., Wolosewick, J.J. (eds.) Scanning
Electron Microscopy inc., AMF OHare (1985)
22. Osada, T., Arakawa, H., Ichikawa, M., Ikai, A.: J. Microsc. 189(1), 43 (1998)
23. Tiribilli, B., Bani, D., Quercioli, F., Ghirelli, A., Vassalli, M.: Ultramicroscopy 102(3), 227
(2005)
24. Favard, P., Carasso, N.: J. Microsc. 97(1), 59 (1973)
25. Hayat, M.A.: Principles and techniques of electron microscopy: biological applications, 4th
edn. Cambridge University press, Cambridge (2000)
26. Reynolds, E.: J. Cell Biol. 17, 208 (1963)
27. Zobel, C., Beer, M.: J. Biophys. Biochem. Cytol. 10, 335 (1961)
28. Van Harreveld, A., Crowell, J.: Anat. Rec. 149, 381 (1964)
29. Magonov, S., Reneker, D.: Annu. Rev. Mater. Sci. 27, 175 (1997)
30. Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K., Watson, J.: Molecular biology of the
Cell. 2nd edn. Garland Publishing, Inc., New York (1989)
31. Renvoize, C., Biola, A., Pallardy, A., Breard, M., Cell Biol, J.: Toxicol. 14(2), 111 (1998)
32. Wild, P., Schraner, E.M., Adler, H., Humbel, B.M.: Microsc. Res. Tech. 53(4), 313 (2001)
33. Hunziker, E., Herrmann, W., Schenk, R., Mueller, M., Moor, H.: J. Cell Biol. 98(1), 267
(1984)
34. Pfeiffer, S., Vielhaber, G., Vietzke, J.P., Wittern, K.P., Hintze, U., Wepf, R.: J. Invest.
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36. Katzmann, J., Odorizzi, G., Scott, D.: Nature Rev. Mol. Cell Biol. 3, 893 (2002)
37. Walther, P., Mller, M.: J. Microsc. 196(3), 279 (1999)
Chapter 4
Structure of the Biological Membrane
(Detection of the Membrane Components
In Vivo)
4.1 Introduction
The ultrastructure of biological membrane is one of the hottest topics in cell

biology. These organelle are involved in a variety of cellular processes such as cell
adhesion, ion conductivity and cell signaling and serve as the attachment surface
for several extracellular structures, including the cell wall, glycocalyx, and
intracellular cytoskeleton. The aim of this chapter is to provide a brief overview
about microscopical techniques which are suitable for the ultrastructural charac-
terization of different kind of cellular membranes at the level of individual mac-
romolecule. Important practical aspects concerning sample preparation, sources of
error and common artefacts as well as recent methodological advances are
discussed.
4.2 Cellular Membrane: Components and Functions
The cell membrane (also called the plasma membrane, plasmalemma or

phospholipid bilayer) is a semipermeable lipid bilayer common to all living
cells. It contains a variety of biological molecules, primarily proteins and lipids,
which are involved in a vast array of cellular processes, and also serves as the
attachment point for both the intracellular cytoskeleton and, if present, the cell
wall [1]. The proportion of membrane proteins/lipids varies between species and
depending to function of a particular membrane. For example: myelin, which
insulates nerve fibers, contains only 18 % protein and 76 % lipid. Mitochondrial
inner membrane contains 76 % protein and only 24 % lipid. Plasma membranes of
human red blood cells and mouse liver contain nearly equal amounts of proteins
(44, 49 % respectively) and lipids (43, 52 % respectively) [1, 2]. The arrangement
of hydrophilic and hydrophobic heads of the lipid bilayer prevents hydrophilic

50 4 Structure of the Biological Membrane
solutes from passively diffusing across the band of hydrophobic tail groups,
allowing the cell to control the movement of these substances via transmembrane
protein complexes such as pores and gates. Specific proteins embedded in the cell
membrane can act as molecular signals which allow cells to communicate with
each other. Protein receptors are found ubiquitously and function to receive signals
from both the environment and other cells. These signals are transduced into a
form which the cell can use to directly effect a response. Other proteins on the
surface of the cell membrane serve as markers which identify a cell to other
cells. The interaction of these markers with their respective receptors forms the
basis of cellcell interaction in the immune system. A thin layer of amphipathic
lipids of the cell membrane are spontaneously arrange so that the hydrophobic
tail regions are shielded from the surrounding polar fluid, causing the more
hydrophilic head regions to associate with the cytosolic and extracellular faces
of the resulting bilayer [1, 2]. This forms a continuous, spherical lipid bilayer
containing the cellular components approximately 57 nm thick. In electron
microscopy the visibility of a plasma membrane is strictly depends on the quality
of protein preservation.
4.3 Practical Aspects of Sample Preparation, Sources

of Error and Common Artefacts
The biological membrane is one of the most dynamical structures: many chemical
and structural rearrangements take place simultaneously and significantly influ-
ence each other. Therefore, conventional chemical fixation, which usually took
from minutes till hours, is not the best choice when the goal of the study is the
investigation of structural aspects which are correlated with dynamical processes
[3]. Cryo-immobilization alone (rapid cooling techniques for thin samples [4],
high-pressure freezing for samples up to 200 lm in thickness [5, 6]) can immo-
bilize biological structures close to their native state because of its high time
resolution for dynamic physiological processes (micro- to millisecond time scale).
Freeze-substitution and freeze-drying (FD) are dehydration techniques by which
the water is gently removed from frozen specimen. Both techniques can serve as a
link between cryofixation and conventional thin sectioning at room temperature.
They are, therefore, hybrid techniques combining the advantages of the low
temperature and the room temperature specimen preparation [7]. For the routine
ultrastructural investigation the freeze-substitution is now the most widely used
procedure. With respect to the structural preservation freeze substitution are more
obscure than pure cryotechniques, such as freeze-etching or cryosectioning.
However cryo sectioning followed by cryo TEM technique is rather difficult to be
used for the routine ultrastructural investigation, as the biological material that is
used for cryo TEM of ultrathin sections, need to be vitrified. At present it appears
nearly impossible to achieve a constant quality of freezing, and vitrification rarely
occurs except in very thin superficial layers, or in objects that contain significant
4.3 Practical Aspects of Sample Preparation, Sources of Error and Common Artefacts 51
Fig. 4.1 TEM micrographs of the cross-section of the antenna of the parasitic wasp Cotesia
glomerata (Hymenoptera: Braconidae). The organism was high-pressure frozen, freeze-substi-
tuted in acetone containing 2 % OsO4
Fig. 4.2 TEM micrograph of

a cross section of C. elegans.
The samples were high-
pressure frozen at the state of
natural necroses, and freeze-
substituted in acetone
containing 20 % Epon/
Araldite mixture
Fig. 4.3 Ultrastructure of nucleolus in correlation with quality of freezing. a sample was frozen
with clear ice segregation pattens bc sample was semi vitrified d sample was vitrified during
freezing
amounts of natural cryoprotectants. In addition, preparation and imaging of

ultrathin cryo-sections is very demanding [8].
4.4 Plasma Membrane by TEM
In spite of high pressure freezing existing already for more than 40 years, and
overall preference (in terms of structural preservation) of cryo fixation over con-
ventional chemical fixation appears to be obvious, the HPF/FS technique still is a
routine processing methods only for a couple of laboratories worldwide. One of the
main reasons responsible is a relatively poor plasma membrane contrast that can
be detected with HPF/FS. The endomembranes, in particular those of the nuclear
envelope, endoplasmic reticulum (ER) and Golgi apparatus, in some cases cannot
be defined at all (Fig. 4.1). This phenomenon is a main reason for the avoidance of
freeze substitution technique for both: histological and 3D tomographical inves-
tigation of the biological samples. Till now a lot of concepts describing the reason
of poor cellular membrane contrast after high pressure freezing have been
4.4 Plasma Membrane by TEM 53
Fig. 4.4 TEM images of the longitudinal section of the cats mite Otodectes cynotis. The
organism was high-pressure frozen, freeze-substituted in acetone containing 20 % Epon/Araldite
mixture (see 2.3). Scale bars equal 100 nm in (a, b), and 300 nm in (c). N nuclei, m mitochondria,
gj gap junction, ER endoplasmatic reticulum. Reproduced from Ref. 8 by permission
presented. Namely, the earliest assumption that the lack of membrane contrast
resulted due to an extraction of lipids by organic solvents during freeze substi-
tution, was contradicted by the fact that after the prolongation of the contact with
OsO4/acetone solution the membrane lipid bilayer becomes clearly visible [8].
Other concept was that the lipids can be extracted during resin embedding, and
was denied later in literature [8]. Numerous publications were devoted to the
enhancement of membrane visibility by adding up to 15 % of water to substi-
tution medium. After numerous debates devoted on the validation of this proce-
dure for structural preservation, Zechmann and colleagues [9???] have proved that
the addition of the water delays substitution to warmer temperatures and causes the
water that is not yet substituted by the FS-media to recrystallize leading to
ultrastructural alterations due to the ice crystal damage.
From our point of view, the membrane appearance depends on many factors:
quality of the freezing (vitrification or semi good freezing quality, when the effect
of the ice crystals can be observed in the nuclear envelope, the most sensitive for
the freezing quality organelle), applied freeze-substitution protocols, different kind
Fig. 4.5 TEM micrographs of the cross-section of the antenna of the parasitic wasp Cotesia
glomerata (Hymenoptera: Braconidae). The organism was high-pressure frozen, freeze-substi-
tuted in acetone containing 20 % Epon/Araldite mixture (see 2.3). Scale bars equal 100 nm in (a,
b), and 500 nm in (c). N nuclei, m mitochondria, ER endoplasmatic reticulum. Reproduced from
Ref. 8 by permission
of chemical fixatives, state of the biological tissue before freezing (was bio-
organism alive before freezing or native proteolyses process was already started)
etc. (Figs. 4.2, 4.3, 4.4, 4.5, 4.6) [8].Direct correlation between membrane protein/
lipids ratio and membrane visibility in TEM after high pressure freezing/freeze-
substitution procedure is almost always evident. Figure 4.1 shows the TEM image
of the neuron system of parasite wasp Cotesia glomerata, which was high pressure
frozen and than freeze substituted according to conventional FS protocol (2 %
OsO4 in water free acetone). As it is evident, the neuron membranes can be
detected as a two separated lines. The visibility of the cell membrane is still
sufficient, but the contrast of double lipid layer is worse compared to neuron.
However, the mitochondria and nuclear membranes are not detectable at all.
Figures 4.2, 4.3, 4.4, 4.5, 4.6 present three different organisms, which were high-
pressure frozen and then epoxy freeze-substitution protocols was applied.
Figure 4.4 shows cross-section of the mite Otodectes cynotis, Fig. 4.5 shows
cross-section of the antenna of the parasitic wasp Cotesia glomerata (Hymenop-
tera: Braconidae), and Fig. 4.6 the human lung fibroblast tissue. All organisms
Fig. 4.6 TEM images of the human lung fibroblast tissue. The cells were high-pressure frozen,
freeze-substituted in acetone containing 20 % Epon/Araldite mixture (see 2.3). Scale bars equal
50 nm in (a, b), 100 nm in (c), and 300 nm in (d). N nuclei, m mitochondria, ER endoplasmatic
reticulum. Reproduced from Ref. 8 by permission
were in the life state before the process of freezing was started, so the problem with
the native proteolysis can be avoided. The quality of the freezing is the only
difference between these three organisms. As it can be clearly seen, the mite was
frozen almost in the vitrified state (we can recognise it from the homogeneous
nuclear state, without any segregation pattern). The contrast of the membranes,
which appear as unstained lines, is significant. Also cytoplasm shows very dense
matrixes. Endoplasmatic reticulum looks like highly ordered organelles. Each
ribosome appears almost identical in shape and equidistant from each other.
Figure 4.5 shows the cross-section of the antenna of wasp, which was frozen
nicely but not vitrified since very small ice segregations can be detected in the
nuclear envelop. The membrane contrast becomes worse in comparison with
Fig. 4.7 TEM ultrathin

section (a) and AFM height
(b) and phase (c) images of
the C. elegans microvilli,
which are localized in the
intestine system. Sample was
high-pressure frozen and
conventionally freeze
substituted. Black arrow
indicates a band of actin
filaments
Fig. 4.4 but is still very strong compared to Fig. 4.6, where almost no membrane
contrast can be observed. On the other hand, the quality of the freezing of the
fibroblast obviously is insufficient (ice segregation almost everywhere in the
Fig. 4.8 TEM micrographs of a similar area of the antennal sensilla placodea of the parasitic
wasp Cotesia glomerata, high- pressure frozen, freeze-substituted in acetone containing 20 %
Epon/Araldite mixture. a Heavy metal stained sample. b Sample after immunocitochemical
analysis for the location of tubulin (the major building block of microtubules)
Fig. 4.9 AFM phase (block face) (a) and TEM (section) (b) images of a similar area of the
antennal sensilla placodea of the parasitic wasp Cotesia glomerata, high- pressure frozen, freeze-
substituted in acetone containing 20 % Epon/Araldite mixture. Cu cuticle
cytoplasm). Nuclear membrane appears in traditional way as two black lines. It is

clear that heavy metal stains have access to lipids bilayer and can intensively stain
them.
Thus, the epoxy freeze-substitution protocol which generally is the best choice
for the protein preservation [8] can not guarantee high membrane visibility of the
sample, protein content of which was damaged due to the ice crystal growth during
the freezing process, or when the native proteolyses of the sample occurs before
Fig. 4.10 AFM phase images of a similar area of the antennal sensilla placodea of the parasitic
wasp Cotesia glomerata, high- pressure frozen, freeze-substituted in (a) acetone containing 20 %
Epon/Araldite mixture and (b) acetone containing 2 % OsO4
freezing. For such samples OsO4 freeze-substitution protocols can be more effi-
cient since it provides good staining quality when the protein state is already
damaged anyway. But when the goal is to obtain an excellent structural preser-
vation and the TEM contrast simultaneously, the epoxy freeze-substitution pro-
tocol applied for the vitrified biosample appears to be the most suitable sample
preparation procedure.
4.5 Plasma Membrane by AFM
The numerous advantages of AFM for the investigation of the macromolecular

content of the high pressure frozen biological samples have been described in
details in chapters 4-6. Here we would like to present a comprehensive analysis of
just two types of cellular membranes. First one is a highly protein packed
microvilli membrane. Different Neuron membranes will the second one.
Microvilli (singular: microvillus) are microscopic cellular membrane protru-
sions that increase the surface area of cells, and are involved in a wide variety of
functions, including absorption, secretion, cellular adhesion, and mechano-trans-
duction. Microvilli are covered in plasma membrane, which encloses cytoplasm
and microfilaments. Each microvillus has a dense bundle of cross-linked actin
filaments, which serves as its structural core. 2030 tightly bundled actin filaments
are cross-linked by bundling proteins fimbrin and villin to form the core of the
microvilli. Microvilli function as the primary surface of nutrient absorption in the
gastrointestinal tract. Because of this vital function, the microvillar membrane is
4.5 Plasma Membrane by AFM 59
packed with enzymes that aid in the breakdown of complex nutrients into simpler
compounds that are more easily absorbed. For example, enzymes that digest
carbohydrates called glycosidases are present at high concentrations on the surface
of enterocyte microvilli. Thus, microvilli not only increase the cellular surface area
for absorption, they also increase the number of digestive enzymes that can be
present on the cell surface. The microvilli are covered with glycocalyx, consisting
of peripheral glycoproteins that can attach themselves to a plasma membrane via
transmembrane proteins [2].
Figure 4.7 represents TEM and AFM images of the C. elegans microvilli,
which are localized in the intestine system. In contrast to TEM, AFM phase image
clearly indicates an advanced macromolecular organization of the membrane
layer. There are protein molecules, which are organized in round microvilli pro-
trusions, and second type of proteins, which creates a continuous radial network
around them. Each microvillus has a dense bundle of cross-linked actin filaments
in the middle, which are also clearly distinguished. It has to be mentioned that the
particular type of enzymes can not be defined by using AFM images alone.
However the correlation of AFM images, obtained from the block face of the HPF/
FS samples along with TEM immunocytochemical analysis can help to clarify the
exact position of each labeled protein molecules.
Figure 4.8 shows the cross-section of the antennal sensilla, labelled against
tubulin, the major building block of microtubules. Positive reaction as indicated by
the presence of gold particle is present inside the outer dendrites. No specific
signal is observed over the tissue section. The immunocytochemical responds is
quite strong in spite of the fact that epoxy resin was used as embedding medium
instead of acrylic resin.
On the other side, microtubules can be clearly distinguished in the AFM phase
image (Figs. 4.9, 4.10). As AFM is a surface oriented technique, the shape of
microtubules were slightly different compare to TEM (instead of continuous solid
protein ring (TEM data), it can be determined as a ring of discrete protein mol-
ecules (AFM)). The usage of osmium tetroxide during fixation make the structure
of microtubules even more grainy, which supports the assumption that the orga-
nelle ultrastructure could be not as dense as it seems from the TEM data
(Fig. 4.10).
References
1. Alberts, B., Johnson, A., Lewis, J., et al.: Molecular Biology of the Cell, 4th edn. Garland
Science, New York (2002)
2. Budin, I., Devaraj, N.K.: J. Am. Chem. Soc. 134(2), 751753 (2011)
3. Ameye, L., Hermann, R., DuBois, P., Flammang, P.: Microsc. Res. Tech. 48(6), 385393
(2000)
4. Dubochet, J., McDowall, A., Menge, B., Schmid, E.N., Lickfeld, K.G.: J. Bacteriol. 155,
381390 (1983)
5. Moor, H.: Theory and practice of high-pressure freezing. In: Steinbrecht, R.A., Zierold, K.
(eds.) Cryo-techniques in Biological Electron Microscopy, pp. 175191. Springer, Berlin
(1987)
6. Mueller, M., Moor, H.: Cryofixation of thick specimens by high pressure freezing. In: Mueller,
M., Becker, R.P., Boyde, A., Wolosewick, J.J. (eds.) The Science of Biological Specimen
Preparation, pp. 131138. (EM, AMF OHare, Chicago (1984)
7. Steinbrecht, R.A., Zierold, K.: Cryo techniques in Biological Electron Microscopy. Springer,
Berlin/Heidelberg (1987)
Chapter 5
Structural and Analytical Chemical
Analysis of the OrganicInorganic
Components in Biomineralized Tissue
5.1 Introduction
Biomineralization is the formation of nanostructured minerals by living cells and

organisms [1]. The biological interest in the field of biomineralization is
obvious- cells, extracellular matrices, transport, signaling, hormone control and
many biomedical implications that have direct bearing on orthopedics, dentistry,
urology etc. Materials scientists study mineralized tissues in order to gain inspi-
ration for developing new synthetic composite materials that are based on natural
systems. Paleontologists and archaeologists are interested in this field because
mineralized tissues make up most of the fossil record and are also major con-
stituents of the archaeological record of our planet [2]. The term biominerali-
sation implies that a mineral phase that is deposited requires or is occasioned by
the intervention of a living organism. This can happen in two basic ways, either the
mineral phase develops from the ambient environments as it would from a
saturated solution of the requisite ions, but requires the living system to nucleate
and localize mineral deposition, or the mineral phase is developed under the direct
regulatory control of the organism, so that the mineral deposits are not only
localized, but may be directed to form unique crystal habits not normally devel-
oped by a saturated solution of the requisite ions. Moreover, the shape, size and
orientation of the crystals may be controlled by the cells involved. The first type of
mineralization was called biologically induced mineralization and the second
organic matrix mediated mineralization. Single-celled organisms and protoctists
such as algae may deposit biologically-induced mineral either intra- or extra
(inter)-cellularly. The majority of eukaryote matrix-mediated mineralization is
extracellular. The variety of structures, as well as the diversity of minerals and
macromolecules that make up mineralized tissues, is amazing [24].

62 5 Structural and Analytical Chemical Analysis of the OrganicInorganic Components
5.2 General Mechanism of Biomineralization
Although detailed mechanism of biomineralization or biocalcification in particular

remain to be elucidated, the basic constructional process of mineralized tissues can
be roughly divided by three general stages: supramolecular pre-organization,
interfacial molecular recognition (templating) and cellular processing.
The initial stage in the formation of biominerals is the construction of an
organized reaction environment before mineralization. In other words, it means a
formation of an organic matrix, which defines and restricts the future mineral
morphology. Two general approaches have evolved in this formation; (1) the self-
assembly of enclosed protein cages and lipid vesicles, and (2) the facilitated
construction of extended protein-polysaccharide network. The former is prevalent
in intracellular biomineralization, whereas the latter is predominant in the extra-
cellular (intercellular) spaces generated by multicellular organisms. Important is
that the organic matrix is not amorphous in the sense that all the atoms are totally
disordered [2]. Three main examples of organic matricesthe amino-polysac-
charide chitin, protein collagen and extracellular polymeric substances (EPS) for
calcium and silica biomineralization contain highly ordered hierarchical structural
organization, in spite of the possible alternatives they provide in chemical nature
and origin (Fig. 5.1) [5, 6].
The second stage in the formation of biological minerals involves the controlled
nucleation of inorganic clusters from aqueous solution. In this process, the organic
matrix provides a framework for the second-order assembly of the inorganic phase.
The supramolecular organization of this matrix consists of functionalized surfaces
that serve as templates for inorganic nucleation. The strongest experimental evi-
dence for the similar local mechanism of the second stage of biocalcification in
both biologically induced and organic matrix-mediated calcification processes is
provided by the detection of silicon in early stages of calcification in the EPS-
attached mineral nuclei from the cyanobacteria suspension (Fig. 5.2) and as a
component of the mineralizing particles and organic matrices in crustacean cuti-
cles (Ligia Italica) [5] (Fig. 5.3), porifera (Demosponges) (Fig. 5.4) [6], in plants
(Morus alba and Ficus microcarpa cystoliths) [7], in insects (Antlion Euroleon
nostras, Neuroptera) as well as in bone, cartilage, and connective tissue of ver-
tebrates (Fig. 5.5) [1, 8, 9]. Glycosaminoglycan-protein matrix (bones, cartilage,
etc.), chitin-protein network (cuticle) and EPS layer (bacteria) [5] all comprise N-
acetyl-glucosamine containing polysaccharides, which can serve as silica-associ-
ation sites. For this reason, the polysaccharide-protein complexes of the biological
calcifying matrices seem to act as local factors that may either prevent the
deposition of inorganic substances or, in the presence of metastable equilibrium of
inorganic ions, catalyze a mineral phase. In the case of biocalcification, silica
aggregates on these polysaccharide-protein complexes and achieves complemen-
tarity between organic and inorganic surfaces Fig 5.6.
The final stage of biomineralization is associated with a variety of construc-
tional processes involving larger-scale cellular activity. In the absence of further
5.2 General Mechanism of Biomineralization 63
Fig. 5.1 Three main

examples of organic
matricesthe amino-
polysaccharide chitin, protein
collagen and extracellular
polymeric substances (EPS)
for calcium and silica
biomineralization contain
high ordered hierarchical
structural organization, in
spite of the possible
alternatives they provide in
chemical nature and origin
cellular intervention, mineral nuclei continue to grow within the confines of their
supramolecular hosts. The resulting particles are constrained in size but have
normal crystallographic structure in morphology.
5.3 High Resolution Microscopical and Analytical Techniques

Used for the Investigation of the Mineral Phase
Usually the mineral precipitates in situ swollen within polymer matrices. While
many biominerals occur in a crystalline form (like molluscan shells consisting of
CaCO3 in its calcitic or aragonitic modification), there are a number of cases where
the biominerals are X-ray amorphous (like in most of structures consisting of
silica: SiO2nH2O) [10]. In the last decade, the attention has increasingly turned to
amorphous and polycrystalline phases that earlier remained mostly undetected due
to a lack of suitable analytical techniques. As most of the biological processes, the
Fig. 5.2 Calcification induced by cyanobacteria. a, e TEM images of cell culture (cells after
cultivation in Z/10 medium with added 8,2 mg/l of phosphorus). b EFTEM RGB map of mineral
particles (RedSi, GreenO, BlueCa jump-ratio images (jri)). c, d EDXS spectra obtained
from the areas EDX 1, and EDX 2, respectively. Yellow color in the EFTEM RGB image
corresponds to a high concentration of Si and O and purple is where Si and Ca co-localize.
Reproduced from reference 5 with permission
mineralisation is also a highly dynamical process which makes the structural

investigation of the cuticle especially challenging. In order to get inside of the
process, the structural investigation has to be performed in close to in vivo state
with the special resolution in a range of nanometers. Correlative AFM and TEM
analyses [11] including supplementary analytical techniques allow one success-
fully localise and indentify both: (I) the mature calcium carbonate deposits
(Fig. 5.7) and (II) the mineral migration inside the newly mineralized tissue
(Fig. 5.3). In this particular case, the usage of each of high resolution microscopy
technique (AFM, TEM (including high resolution TEM (HRTEM)), elemental
5.3 High Resolution Microscopical and Analytical Techniques 65
Fig. 5.3 Calcification of Ligia italica cuticle. a TEM image of exocuticle; b EFTEM RGB map
(RedSi, GreenO, BlueCa jri). e AFM phase image of the similar area of exocuticle. c,
d EDXS spectra obtained from areas EDX 1, and EDX 2, respectively. Yellow color in the
EFTEM RGB image corresponds a high concentration of Si and O and purple is where Si and Ca
co-localize. Reproduced from reference 5 with permission
mapping by energy filtering TEM (EFTEM) and electron energy loss spectroscopy
(EELS) is necessary in order to give a comprehensive description of the sample.
TEM provided information about the general organization of mineral distribution
at the tissue, Ca, O, Si can be localized by EFTEM elemental mapping and EDX in
STEM mode. The thickness and the morphology of the mineralized areas near the
surface as well as the size and the distribution of mineral clusters can be char-
acterized by AFM. Also HRTEM imaging is necessary in order to clarify a
crystalline form of both the mature CaCO3 deposits and mineral clusters in earlier
state of mineralization which are completely amorphous.
Fig. 5.4 Identification of mineral components within clublike constructions. a The SEM image
of a mechanically disrupted clublike spicule shows that the spines each possess a nucleus that is
covered by silica layer. b Magnification of (a). Elemental mapping (c) and EDX analysis
(d) show the presence of calcium as the main component of this nucleus. e Photoemission spectra
of club-formed spicule showing the presence of two kinds of silicon oxides. f X-ray absorption
spectra showing that calcium carbonate in the form of calcite is the second mineral component
present within the spicules. Reproduced from reference 6 with permission
5.4 Correlative AFMTEM
Chitin is a common constituent of the arthropod exoskeleton in general, including

insects, crustaceans, chelicerates, and myriapods. The most characteristic feature
of the chitinous cuticle, which is a biological nanocomposite material, is its strict
hierarchical organization, which reveals various structural levels: First, at the
molecular level is the polysaccharide chitin itself. Its antiparallel alignment forms
5.4 Correlative AFMTEM 67
Fig. 5.5 a SEM micrographs

and b EDX analysis of a
cross-section of (A1, B1)
porous b-TCP, (A2, B2)
50 % b-CS, (A3, B3) 80 %
b-CS, (A4, B4) b-CS after
implantation for 4 weeks.
Reproduced from reference 8
with permission
Fig. 5.6 Light microscopy investigation of Ca2+ oxalate crystals and demonstration of the use of
LC-PolScope image analysis. ac Begonia rex Ca2+ oxalate crystals (indicated by arrows) in
bright field contrast (a) LC-PolScope retardance mode (b) and LC-PolScope orientation mode (c).
The retardance mode allows calculation of birefringence (double refraction) of a crystal. The
orientation mode gives information with respect to the orientation of the light passing through the
slow optical axis of a birefringent crystal. (d and e), Allium spec. Ca2+ oxalate crystals analyzed
in LC-PolScope retardance mode (d) and vector overlay mode (e). The vector represents the slow
optical axis of a birefringent crystal. Reproduced from reference 7 with permission
alpha-chitin crystals. The second structural level is the arrangement of 1825 of

such molecules in the form of narrow and long crystalline units, which are
wrapped by proteins, forming nanofibrils of about 25 nm in diameter and about
300 nm in length. The third step in the scale consists of clustering of these
nanofibrils into long chitin-protein fibrils of about 50300 nm in diameter. These
chitin-protein fibers form a planar woven and periodically branched network
(chitin-protein layers). The spacing between the fibers is filled up with proteins and
biominerals of microscopic and nanoscopic size [12, 13].
Until now, the structural organization of the native chitin has been revealed by
various microscopic and analytical techniques with different degrees of resolution.
Usually, X-ray diffraction, TEM, and SEM at high resolution have been used to
determine the orientation of fibers. Raman or infrared analysis is the most popular
method to determine the mineral composition (Fig. 5.8); while the protein content
of the cuticle has been examined by protein extraction followed by two-dimen-
sional gel electrophoresis, immunocytochemistry etc. [10]. Although each of the
above mentioned techniques brings a big impact to the understanding of chitin
Fig. 5.7 Analytical study of the CaCO3 deposits that are localized in the exocuticle area of the
mature cuticle of Ligia italica. a AFM phase image of the exocuticle, (a (insert)) organization of
CaCO3 clusters at higher magnification, b correlative TEM image of the same area, c,
d corresponding RGB images of the same area of exocuticle: redCa, greenO, yellowSi.
Scale bars: 200 nm in (a), 100 nm in (a (insert))
structure, the original organization of the native chitin (polysaccharide chains,

proteins, and biominerals in there original places within the cuticle), can not be
visualized by any technique till now. The reason is that each component requires
special experimental conditions and as a consequence a different measuring
technique which is usually optimal for one and not suitable for other two chitin
constituents. For example, demineralisation and protein hydrolysis is required in
order to investigate polysaccharide chitin structure by x-ray diffraction. Precise
protein analysis usually done using proteins solutions extracted from the rest of the
cuticle. For the investigation of the mineral content of the chitin by Raman or
infrared analysis, the sample has to be dried, so the native hydrated shape of the
proteins is completely collapse.
Fig. 5.8 Electron probe microanalysis (EPMA) of sagittally cleaved and microtome polished
surfaces of the mineralized high-pressure frozen and freeze-dried tergite cuticles of Porcellio
scaber (ad) and Armadillidium vulgare (eh). (a and e) SEM image and elemental X-ray
spectrum indicate the presence of C, O, Mg, P and Ca. The aluminium peak is due to the use of
the conductive glue at the sides of the specimen. Spectral maps and line scans are shown for
calcium (b and f), magnesium (c and g) and phosphorus (d and h). The arrows in (a) and
(b) indicate the position from which line scans were recorded. Reproduced from reference 10
with permission
Fig. 5.9 AFM amplitude images of the HPF mite Otodectes cynotis. a image shows structural
preservation of the tissue of the sample which have been embedded in LRWhite resin, b the
sample which have been embeded in epoxy resin
Fig. 5.10 Correlative AFM phase (block face) and TEM (last ultrathin section) images of a
cross-section of HPF/epoxy FS cuticle of mite Otodectes cynotis. EDX and EELS spectra
represent the chemical composition of the selected area. Scale bars are 250 nm. Phase variation:
05. Reproduced from reference 14 with permission
The universal method for the investigation of the chitin structure in it native like
state by using a correlative AFM-TEM analysis [14] is presented here. The
advantage of this method is that all three components could be preserved simul-
taneously at the sample using high-pressure freezing/epoxy freeze-substitution
methods and then visualised using both (AFM and TEM) high resolution micro-
scopical methods. AFM is applied for the visualisation of protein-chitin fibrils,
protein-mineral complexes and there mechanical properties, while conventional
TEM serves for the description of sample organisation in general. The strongest
feature of the AFM-TEM correlative method is that the information can be obtained
from the same particular specimen area (same organelle, same chitin-protein fibril
etc.). The only requirements are an appropriate sample preparation procedure
(fixation, embedding) and optimal AFM and TEM measurement conditions.
5.5 Sample Preparation Procedure
The ultrastructural appearance can be deliberately influenced with the standard

aggressive chemicals like osmium tetroxide which are conventionally used during
Fig. 5.11 Organic matrix of the cuticle. a TEM images of the areas of exo- and endo-cuticle.
b EFTEM carbon jri. e, f AFM height images of conventionally fixed cuticle and cuticle after
proteolytic treatment. c, d EDXS and EEL spectra obtained from areas EDX 1, and EELS 1,
respectively. Cu L alpha peak is originating from the Cu grid bars. Reproduced from reference 5
with permission
chemical fixation or freeze-substitution procedure. Such protocols usually partially

or even completely degrade cytoplasmic and membrane proteins. Other disad-
vantage of the OsO4 usage is that osmium containing samples can not be used for
the TEM analytical investigation. Therefore, if AFM-TEM correlative microscopy
is concerned, than the optimal sample fixation would be a high-pressure freezing/
epoxy freeze substitution [14]. High pressure freezing provides the best structural
preservation of the sample. Epoxy stabilisation during freeze-substitution and
embedding guaranties the highest quality of the AFM images of the cuticle. The
5.5 Sample Preparation Procedure 73
usage of hydrophilic resins (e.g., most acrilates and methacrylate based resins)
which are spread widely because of their high suitability for on-grid immu-
nolocalization do not provide a sufficient structural contrast nether for cytoplasmic
components nor for chitin ultrastructure (Fig. 5.9).
5.6 Polysaccharides-Protein Filaments
It appears to be clear that comprehensive understanding of the role of proteins in the

native chitin can be obtained not only by investigation of the extracted proteins but
also by structural analysis of the distribution of the protein matrix within the cuticle
which is preserved as accurate as possible. Figure 5.10 represents the complemen-
tary couple of images of the cuticle of the mite. The sample has been high pressure
frozen/epoxy freeze substituted and epoxy embedded. As it has been mentioned
above, this method provides the most accurate (after high pressure freezing/cryo
sectioning) protein preservation of the biological materials. The ultrastructure of
cuticle is manifested differently in TEM and AFM images. TEM image shows only
those polysaccharide-protein fibers which can be stained properly and which can be
visualized as separate constituents of sample volume in 2D projection of near 50 nm
thick section layer (TEM projection issue). AFM phase image contains both: the
oriented polysaccharide fibers (nicely oriented patterns of thin filaments) and the
protein macromolecules, which appears as grained structure around and between the
chitin fibers. Also phase image depicted the different stiffness of the proteins layers
within the cuticle: each two neighboring layers contain the soft area (dark phase
contrast) in between (Fig. 5.10). As the mite cuticle do not contain minerals in the
concentration which can significantly influence the stiffness of layers (EDX spectra
obtained from the dark and bright regions show almost the same chemical content),
one can suggest that the different stiffness can be attributed mostly to the different
protein composition of the dark and bright regions.
It has to be mentioned that the protein distribution within the biological chitin is
also not homogeneous in terms of large scale (micrometers). This effect can be
detected perfectly by AFM phase image with very high resolution [14]. TEM
micrograph of the longitudinal section of mite cuticle indicates quite uniform
fibrils thickness as well as there orientation patterns. In contrast to TEM image,
AFM phase images show that the chitin-protein fibrils have a great variety of
orientation patterns and also an essential difference in the size distribution.
Because of projection issue and low EM contrast of light elements (C, O, H), such
information is usually missed in TEM data.
5.7 Analytical TEM
Isopod crustaceans are convenient models to study calcification due to frequent

molting and a thin, soft cuticle, which enables microscopic analyses of intact, non-
decalcified samples. The mineral part of the isopod cuticle consists of crystalline
calcium carbonate in the form of calcite, amorphous calcium carbonate and

amorphous calcium phosphate [15]. To obtain detailed information on ultra-
structure, chemical composition, and mechanical properties of the calcifying
cuticle on the nanometer scale, TEM imaging and chemical analyses (EFTEM,
EDXS, electron energy loss spectroscopy (EELS)) of ultrathin sections were
combined with atomic force microscopy (AFM) imaging of the block face in the
same sample. A significant amount of silicon has been demonstrated within this
matrix by EDX. EFTEM (Fig. 5.11) showed that the highest concentrations of
silicon are present at the interface of matrix and chitinous bundles. AFM phase
imaging has revealed numerous hard aggregates, distributed at the matrix-bundles
interface (Fig. 5.11c). These aggregates also contained calcium and phosphorus, in
contrast to the matrix, where these elements were not detected. The shape of the Si
L23 ionization edge in the EEL spectra (Fig. 5.11d) obtained from the matrix is
specific for hydrated silica (SiO2nH2O) [16]. The smooth layer of the exocuticle
consists of hard substances and displays oblique layers in AFM height images
(Fig. 5.11e). To clarify the nature of the matrix, samples were subjected to
deproteinization (Fig. 5.11f), which resulted in disorganization of the exocuticular
matrix. The clusters in the damaged matrix displayed sharp angles and hexagonal-
prism shapes, characteristic for a hard-mineral-containing phase. The fact that the
noncalcified part of the matrix was destroyed after deproteinization in contrast to
the already calcified one strongly supports the conclusion that proteins are an
important constituent of this organic matrix. In previous studies calcite was
exclusively detected in the exocuticle in crustaceans, mostly restricted to the thin
outermost layer of the exocuticle, while in the endocuticle amorphous calcium
carbonate was localized [10, 16]. However, this silicon-containing mineralizing
proteinaceous matrix is located exactly in the area which is reported to contain
calcite in the mature cuticle.
References
1. Carlisle, E.M.: Sci. Total Environ. 73, 95 (1988)

2. Veis, A.: Rev. Mineral Geochem. 54(1), 249 (2003)
3. Lowenstam, H.A.: Science 211, 1126 (1981)
4. Lowenstam, H.A., Weiner, S.: In biomineralization. Oxford University Press, New York
(1989)
5. Matsko, N.B., Znidaric, N., Letofsky-Papst, I., Dittrich, M., Grogger, W., trus, J., Hofer, F.:
J. Struct. Biol. 174(1), 180 (2011)
6. Ehrlich, H., Brunner, E., Simon, P., Bazhenov, V.V., Botting, J.P., et al.: Adv. Funct. Mater
21(18), 3473 (2011)
7. Bauer, P., Elbaum, R., Weiss, I.M.: Plant Sci. Vol. 180(6), 746 (2011)
8. Wang, C., Xue, Y., Lin, K., Lu, J., Chang, J., Suna, J.: Acta Biomater 8(1), 350 (2012)
9. Carslie, E.M.: In silicon biochemistry, Ciba Foundation Symposium 121 Wiley, Chichester
(1986)
10. Hild, S., Marti, O., Ziegler, A.: J. Struct. Biol. 163(1), 100 (2008)
11. Matsko, N.: Ultramicroscopy 107, 95 (2006)
References 75
12. Vincent, J.F.V.: Structural biomaterials. Princeton University Press, NJ (1990)

13. Raabe, D., et al.: Acta Mater 53, 4281 (2005)
14. Matsko, N., Letofsky-Papst, I., Znidaric, N., trus, J., Grogger, W., Hofer, F.: Imaging
Microsc. 12(2), 40 (2010)
15. Dillaman, R., Hequembourg, S., Gay, M.J.: Morphol. 263, 356 (2005)
16. Muller, D.A., Sorsch, T., Moccio, S., Baumann, F.H., Timp, G.: Nature 399, 758 (1999)
Chapter 6
Cellular Dynamics (Protein Transport,
Mineralization In vivo)
6.1 Introduction
A living cell can be described as a highly dynamical organism where different

kinds of physiological processes may happen simultaneously. These processes take
place at different length scales and are strongly influenced by each other. In order
to circumvent the complexity and the dynamics of biological systems, so far many
of the processes have been studied mostly in vitro through the use of
two-dimensional molecular assemblies as model systems [1]. Such an approach is
very useful for monitoring physiological processes in vitro, but it hardly can be
applied for an accurate description of the native process in a living cell or tissue,
since it represents a multicomponent chemical context where each of cellular
constituents can be involved in more than one process simultaneously. True
investigations of the cellular dynamics in vivo are challenging and complicated but
paramount at different levels from the cellular metabolism of each cell in particular
until physiological processes on the macro scale like tissue formation, degenera-
tion, or geo-mineral formation involving bio-mineralization.
Until now there exist two main approaches to resolve fast structural changes in
the cell. The first one is life imaging in vivo. Confocal and fluorescence micros-
copy [2] and in situ investigations of biological samples by environmental scan-
ning electron microscopy (ESEM) [3] or atomic force microscopy (AFM) [4] are
the main techniques which are used. However, the information about cellular
dynamics obtained by these techniques usually is limited by the necessity to
introduce artificially designed dyes into the alive cell in the case of fluorescence
microscopy, or by the necessity to work with the sample under low vacuum
condition (ESEM). In addition in ESEM and/or AFM experiments the dynamical
events in the cell can be observed only indirectly, because there is no possibility to
cut the cell in order to get inside and still keep it alive.
A second approach makes use of cryo methods. The uniqueness of cryo studies is
related to the fact that high pressure freezing which is used for the fast fixation of

78 6 Cellular Dynamics
Fig. 6.1 Protein transport within the nuclear porous. a sample was vitrified, b Semi vitrified
sample, and c sample which was frozen with ice segregations patterns. Black arrows indicate the
area of nucleus
hydrated samples provides a high cooling rate, which guarantees a high time resolution
for dynamic cellular events, i.e. a rapid (* 0.05 s) stop of all physiological processes
[5]. As most of the dynamical events within the cell happen in the range of ms to ls, the
subsequent cryo microscopy of high pressure frozen samples allows one to come very
close to their real native state, which is one of the ultimate goals of microscopical
investigations of biological objects. In case when sample is truly vitrified, the
dynamical evens can be easily detected even by conventional TEM. (Fig. 6.1)
On the other hand, it is not enough just to preserve the internal structure of the
sample in the best way, it is also crucially important to properly detect it. There are
two main microscopic techniques that allow obtaining structural information of an
object in the nanometer range: (1) transmission electron microscopy (TEM)
including scanning TEM (STEM), and (2) scanning probe microscopy (SPM)
including atomic force microscopy (AFM). TEM is nowadays the most widely
spread technique used for the investigation of biological samples, although the low
contrast of cellular constituents, the necessity to use a two-dimensional projection
of the sample volume, and the issue of electron beam damage limit the material
range and abilities of the technique. Alternatively, the AFM has a nondestructive
character, and since it is primarily a surface characterization technique, radiation
damage of the sample surface can be avoided [6]. The possibility to obtain
information about the location, architecture and mechanical properties of macro-
molecules or polymer chains in the nanometer range directly from the surface of
the section or block face makes this technique extraordinary useful for the
investigation of local changes within the sample that take place during dynamic
processes as well as the whole ultrastructure in general.
6.2 Detection of the Macromolecular Content of a Cell

by AFM and its Interpretation Using Complementary
AFMTEM Analysis
The interpretation of new results in any subdivision of microscopy in life sciences

is currently based on our knowledge about cellular ultrastructure that has been
obtained by TEM of ultrathin sections of biological material. Most of the
6.2 Detection of the Macromolecular Content of a Cell 79
Fig. 6.2 shows a cross-section of high pressure frozen (HPF) and then epoxy freeze substituted
(FS) C.elegans. Cryo-immobilization preserves biological structures in a state very close to
native, because of its microsecond time resolution for dynamic physiological processes in
contrast to conventional chemical fixation that takes seconds to hours Reproduced from the Ref.
[13] with permission
Fig. 6.3 Biphasic molting of Ligia italica. Different states of mineral resorption which can be
observed within particularly the same sample block face. Black arrows indicate spherules with
different hardness and chemical composition
bio-macromolecules (proteins, polysaccharides, nucleic acids) as well as polymer/

copolymer chains mainly consist of light elements (C, H, O, N), which scatter the
incident electrons rather weakly. Consequently these compounds can be detected
on TEM micrographs only as a grayish background [7]. It has to be mentioned that
most of the dynamical events within the cell are regulated by specific proteins and
therefore the comprehensive ultrastructural description of a dynamical process
strictly requires a high resolution imaging technology, which can provide infor-
mation about the protein content of a cell. Staining of the sample can considerably
improve the image contrast, but the heavy metal salts, which are used for such
purpose, have a certain size and can penetrate deep into sample only when the
interaction with the macromolecules or the chain matrix is relatively weak. Thus,
Fig. 6.4 Biphasic molting of Ligia italica: spherules structure. a AFM phase image of the
sample block face b TEM image of the similar area of the sample
only the structures, which react with the staining agents, and which can be reached
by the staining agents are detected [8]. Consequently, relatively big cellular
organelles, membranes, protein filaments, and nucleic acids are clearly observed,
but many proteins in the cytoplasm are practically invisible. For cryo TEM of
ultrathin sections the situation concerning macromolecule detection is even worse,
as cryosections cannot be stained at all. Since all the cellular proteins have a
similar scattering ability, they cannot be resolved in a 2D projection at all.
Therefore, from the TEM image of both cryo sections (frozen hydrated samples) or
resin sections (high pressure frozen and freeze substituted samples), it is almost
impossible to estimate the macromolecule state of the biological objects. This is
due to the fact that the high stainability of the cellular membranes can equally be a
sign of membrane protein degradation or indicate a well preserved membrane,
which natively contains very low proteins like neuron membranes. AFM, on the
contrary provides information about the cell constituents that are distributed on the
surface of the block face. Our previous studies clearly demonstrate that AFM
represents a powerful tool for the estimation of the cellular macromolecular
content, including its distribution and architecture [7]. Equally, AFM is the best
technique for the estimation of structural preservation of biomaterials embedded in
epoxy resin in terms of quality of freezing and preservation of the ultrastructure
during freeze substitution [9]. Therefore, the usage of AFM as a complementary
microscopic technique to TEM can be extraordinary useful for the investigation of
dynamical processes within the cell, where the key role is played by macromo-
lecular complexes. Especially useful as well as challenging to perform is the
investigation of exactly the same place of the specimen which provides the most
adequate correlative AFM/TEM analysis.
In the TEM image (Fig. 6.2b), some areas of the nuclear membrane look
slightly dissolved (see arrows). From this information, it cannot be clearly dis-
tinguished between three possibilities: (1) Such areas correspond to nuclear pores
6.2 Detection of the Macromolecular Content of a Cell 81
Fig. 6.5 Chemical composition of spherules. a Conventional bright field image of ultrathin non
stained section b corresponding EFTEM maps: red color corresponds to the phosphorus
distribution, greencalcium distribution
involved in the transport of macromolecule across the nuclear envelope [10]; (2)
The low contrast of these areas is a staining artifact; (3) The membrane orientation
was not parallel to the electron beam.
The AFM image (Fig. 6.2a) shows that these areas correspond to almost solid
bridges between the cytoplasm and the nucleoplasm, of macromolecular nature
[7]. It should be noted that the membrane transport is a highly dynamic process
(ref) and proper detection of the proteins which are involved in this process
requires additional biophysical methods like immunocytochemistry, cryo TEM
including cryo analytical TEM (elemental mapping by energy filtering TEM
(EFTEM), energy dispersive x-ray spectroscopy (EDXS) and electron energy loss
spectroscopy (EELS)) etc, as well as an accumulation of a big statistical database.
Hereby, the direct imaging opens up new horizons for the investigation of dynamic
membrane processes at the level of individual macromolecular components.
6.3 Spherules Involved in Elaboration of Crustacean Cuticle

During the Molt Cycle: A Correlative TEMAFM Study
Frequent biphasic molting is a unique feature of terrestrial isopod crustaceans. In

intramolt animals the interior part of the body is still covered with both cuticles
(Fig. 6.3), while the old cuticle of the posterior part is shed and the new cuticle
calcifies. The intramolt stage is convenient for structural analysis of pre and
postecdysal cuticles of the same animal. Resorption of minerals from the cuticle
and storage of calcium in ventral parts of the body during premolt is an important
adaptation of isopod crustaceans to terrestrial lifestyle. This is also a highly
dynamical process. Figure 6.3 shows different states of mineral resorption which
stained section b EELS spectra from the area, presented in a, and c corresponding EDX spectra
from the same area
stained section b corresponding RGB EFTEM maps: red color corresponds to the silica
distribution, green to the carbon distribution, and blue to the calcium distribution
can be observed within particularly the same sample block face. In Porcellio
scaber the sternal deposits are composed of amorphous calcium carbonate and
calcium phosphate. Calcium spherules are formed by aggregations of nanogranules
in aspecialized aggregation zone (Figs. 6.4, 6.5, 6.6, 6.7) [11].
In premolt animals numerous spherules surrounded by electron dense ecdysal
matrix are formed by the resorption of material from disintegrating lamellae of the
old endocuticle (Figs. 6.4, 6.5). The size of the spherules composed of concentric
6.3 Spherules Involved in Elaboration of Crustacean Cuticle 83
layers with electron dense deposits is about 0.5 lm. Fine electron dense deposits
are also present in the pore canals of the new exocuticle, at the microvillar pro-
jections of epithelial cells and in the intercellular spaces. EFTEM map shows the
calcium and phosphorus distribution within both the spherules and ecdysal matrix.
The AFM micrographs of the same sample show that the spherules are loaded with
harder material from the ecdysal matrix, as progressing from the basal lamellae of
the old cuticle through ecdysal space towards the surface of the new cuticle
(Fig. 6.4). The ecdysal matrix close to the old cuticle exhibits harder texture
compared to softer texture of the rest of the matrix. Spherules attached to the new
epicuticular layer exhibit a hard granular texture. The granular material is con-
centrated in the intercellular spaces and in the exocuticular pore canals and it is
finely dispersed in the cytoplasm and nuclei of epithelial cells. The chemical
content of each particular area of sample can be detected by ATEM. EDX in this
case shows the presence of Ca, P, C, O, N, and Si. EELS spectra provide the
information about chemical state of the minerals. Here have to be mentioned that
for some elements like for example Si and P, the EFTEM mapping will require an
additional check out, due to the superimposed Si and P ionization edges. Simply
telling, when such situation take place, the EFTEM map can show a presence of
both elements, when in reality only one is there. Therefore EDX spectra in
addition to EFTEM can bring the clarity to this problem.
As a conclusion the combination of ATEM and AFM analyses enables us to get
the information about the detailed ultrastructure, composition and mechanical
properties of the sample in the nanometer range. In addition, this approach is very
convenient for deciphering dynamic processes like molting, where the structural
and physicochemical properties change over short distances.
References
1. Pouget, E.M., et al.: Science 323, 1455 (2009)

2. Pawley, J.B.: Handbook of Biological Confocal Microscopy, 3rd edn. Springer, Berlin (2006)
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4. Drake, B., et al.: Science 243, 1586 (1989)
5. Mueller, M., Moor, H. in: The Science of Biological Specimen Preparation, ed. by M.
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9. Matsko, N., Mueller, M.J.: Struct. Biol. 152, 92 (2005)
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11. Fabritius, H., Ziegler, A.J.: Struct. Biol. 142, 281 (2003)
12. trus, J., Znidaric, N.,Tusek Znidaric, M., Grogger, W., Hofer, F. Matsko, N., Pabst, M.A.,
Zellnig, G. (Eds.) MC2009, Vol 2
13. Matsko, N., Letofsky-Papst, I., Znidaric, N., trus, J., Grogger, W., Hofer, F.: Imaging &
Microscopy 12(2), 40 (2010)
Chapter 7
Tomography of the Hydrated Materials
7.1 Introduction
The preservation of native structures of soft polymers and biological organisms

during sample preparation and microscopic study is the ultimate requirement for
comprehensive analyses at the level of individual macromolecules. A combination
of low temperature techniques such as cryo sectioning (cryo ultramicrotomy [1]
and cryo focus ion beam [FIB] milling [2]), followed by high-resolution cryo
microscopy study (cryo transmission electron microscopy, cryo TEM [3, 4]), has
proved to be the most powerful approach available so far for ultrastructural
investigation of the bulk of soft materials. Over the last few years, interest in cryo
TEM imaging technology has led to major progress in both the 3D reconstruction
technique (by cryo tilt-series-based tomography [5] which allows for solving the
projection limitation), as well as in the enhancement of image contrast (by phase
contrast electron microscopy [6]). The necessity for the use of high vacuum
conditions during TEM analysis, however, poses certain problems as sample mass
is lost, which is especially severe concerningfrozen hydrated systems [7]. Despite
this, almost all soft organic materials are prone to electron and ion beam damage
which results in an unfavorable structural reorganization [7, 8]. Both points
mentioned above may lead to a partial or even complete loss of sample nativity
during observation. An atomic force microscopy (AFM) [9] is a surface charac-
terization technique [10] which, in contrast to TEM, can be considered non-
destructive for the analysis of soft matter. The reason for this phenomenon is that it
requires neither electron or ion beams, nor high vacuum conditions for operation,
and therefore excludes partial freeze drying and radiation damage of the sample
surface. Information about the location, architecture and mechanical properties of
nano-sized macromolecules or polymer chains can be obtained directly from the
surface of the unstained block face [11, 12] which is free from sectioning arte-
factseven at low temperatures [13]. This technique is thus particularly useful for
the investigation of both local changes within a sample occurring during dynamic

86 7 Tomography of the Hydrated Materials
processes, and the entire native ultrastructure in general [11, 12]. In this chapter,
both topographical techniques: cryo TEM tilt serious based tomography and cryo
serial section AFM tomography are presented. As it was already mentioned before,
these two techniques do not contradict each other, but rather serves as a
complementary to each other.
7.2 Cryo TEM Tilt Serious Based Tomography
In electron tilt-series-based tomography, 2D images of a specimen are acquired

with viewing from different angles and then synthesized into a 3D mass density
map. The specimen holder is tilted incrementally around an axis perpendicular to
electron beam, e.g. from -65 tilt to +65 tilt with 1 increment, and images are
taken for each position. Before computation of the tomogram, the projection
images must be mutually aligned within a common frame of reference. TEM tilt-
series-based tomography is a very promising technique for obtaining high
resolution structural data of macromolecules, small filler particles and defects, but
may not be applicable when larger volumes are needed to be reconstructed, since
section thickness is limited to around 1 lm [14]. Ultra-rapid freezing immobilizes
biological samples and ensures that they are in a close-to-native state during
imaging [15]. Compared with conventional electron microscopy (EM), which
involves chemical fixation, dehydration and staining, cryogenic sample prepara-
tion methods have greatly improved our understanding of the biological ultra-
structure and macromolecular organization within a cellular context [16]. The
technique has its own limitations, however, which must be recognized and
addressed. One of the major difficulties in imaging frozen-hydrated biological
samples is their sensitivity to the electron beam; therefore, a limited dose must be
used during image acquisition to reduce the potential of ultra-structural damage to
the sample. It has been well documented that beam damage, in the form of vitreous
section flow, during image acquisition [17, 18] significantly limits how one
should distribute electron dose during single-projection imaging and also during
tilt series acquisition. The result is a low-dose imaging method with an increased
amount of statistical noise due to inadequate sampling that limits both the
observable cellular detail and the subsequent procedures for the alignment of
multiple examples of the same biological structure (Fig. 7.1). Confident recogni-
tion of macromolecular complexes within the three-dimensional (3D) snapshots of
cells imaged with low electron doses remains a challenge, partially due to the low
signal-to-noise ratio caused by low-dose imaging. Non-isotropic sampling and the
absence of an in vivo labeling technique are additional important problems. Only
large macromolecular complexes and cellular components [1925] can be recog-
nized with confidence. Artifacts induced by the process of sectioning (knife marks,
crevasses and compression) have been described in detail and must be taken into
consideration. Small macromolecular complexes can be visually recognized based
on their size and distribution. However, the true identity of these protein
7.2 Cryo TEM Tilt Serious Based Tomography 87
Fig. 7.1 a An image from a medium magnification montage EM grid map (using a Tecnai F20
[FEI, Eindhoven] EM at 1409 nominal magnification) showing a representative area of
Saccharomyces cerevisiae (denoted by the Y) and NRK (outlined with black dotted line) cells.
The cells were mixed together, high-pressure frozen and vitreous cryo-sectioned. b A 10-nm slice
from a reconstructed tomogram of an NRK cell. The letter M represents a mitochondrion and N
the nucleus. b0 A selected area diffraction pattern from b indicating that the sample is vitreous.
Scale bars = 100 nm. Reproduced from Ref. [26] with permission
complexes remains subjective until a suitable labeling, and correlative approach or

template matching procedure is developed for electron cryo-tomography. One
major limitation is the lack of a green fluorescent protein-like label for EM.
Therefore, localizing individual protein complexes in cells remains a challenge.
There have been recent advancements, though, that correlate cryo-light
microscopy images with cryo-EM images to mimic protein labeling, or correlative
light-electron microscopy. This technique is rapidly developing and may replace
the need for a green fluorescent protein-like EM marker in the future [26].
Figure 7.2 represents an example of 3D reconstruction of ribosome by 3D tilt
serious based tomography.
7.3 Cryo AFM Serial Section Tomography
Cryo atomic force microscope system (SNOTRA) consists of a specifically

designed cryo-AFM which is mounted directly within the cryogenic chamber of an
ultramicrotome. The combination of these devices allows an analysis of soft and
frozen hydrated materials immediately after sectioning at room temperature or
cryo conditions. Moreover, SNOTRA can also be employed for investigating
Fig. 7.2 Multi-resolution mapping of an 80S S. cerevisiae ribosome density map obtained from
tomographic reconstructions of vitreous cryo-sections. a 80S ribosome density map obtained
from tomography of vitreous cryo-sections. The small (40S) and large (60S) subunits are denoted
along with representative ribosomal landmarks including the head region (h), beak (bk), shoulder
(Sh), base (b), stalk (St), and stalk base (SB). a0 180 rotated view from a that shows the L1
protuberance (L1). b A high resolution single-particle 80S S. cerevisiae ribosome obtained from
single-particle cryo-EM analysis is fit into the density map in a and a0 . c The theoretical X-ray
crystallographic map fit into the density map from a. Green color represents the 40S small
subunit and blue color is the 60S large subunit. Reproduced from Ref. [26] with permission
thermotropic dynamic processes in soft materials, and for serial section 3D

tomography at a wide range of temperatures (50120 C).
The cryo-AFM system employs chemically etched tungsten tips attached to a
quartz tuning fork as an AFM probe [2729] (Fig. 7.2). At this configuration,
several AFM measuring modes such as amplitude feedback as semi-contact AFM,
phase imaging, semi-contact feedback-error mode [29], and shear force mode
(parallel attachment of the tip to a tuning fork prong) are available [30]. The level
of mechanical noise between the tip and the sample is less than 0.1 nm RMS under
cryo conditions, which is a satisfying value for AFM measurements, considering
that the system operates in flowing LN2 cooling gas environment. The spatial
resolution of the cryo-AFM depends mostly on the probe tip sharpness; a level of
510 nm can be achieved (Fig. 7.3).
The cryo-AFM unit moves together with the arm of the cryo-ultramicrotome
(Fig. 7.2). The probe approaches the surface by means of a stepper motor when the
arm is driven to the upper position. This motor is mounted on the front of the
cryo-chamber outside of the cooled zone and moves the probe carriage by a system
of levers and flat spring guides. After measuring, the probe is removed from the
sample surface in the same way. It is thereafter possible to proceed with the next
7.3 Cryo AFM Serial Section Tomography 89
Fig. 7.3 Instrumentation set up of SNOTRA. a Schematic 3D-model of the cryo-AFM layout.
b Photograph of the cryo-AFM system. The cryo-AFM unit is placed instead of a conventional
sample holder of the cryo-ultramicrotome. The sample investigated is glued or frozen to a nickel
support plate (1), which is fixed magnetically on a horizontally oriented piezotube scanner (2)
with a scan range of 50 9 50 9 5.0 lm at room temperature. The scanner is mounted on a base
platform with guide notches, on which a moveable carriage (3) with an AFM probe (4) is
installed. The carriage may be moved along the guide notches in order to approach the sample.
The base platform of the cryo-AFM unit has an opening below the sample for the diamond knife
(5) of the cryo-ultramicrotome, with which one can make a section of the sample. After the
section the arm is driven to the upper position and the opening in the base platform is closed by a
flat mica cover. Another cover made of plastic closes the base platform and a measurement zone
from the top. It helps to minimize gas flows in the measurement zone, which may disturb the
measurements. c Diamond ultramicrotome knife with customized shape for cryo-AFM (Diatome
AG). d Tuning fork with chemically etched tungsten wire tip attached (SEM micrograph)
Fig. 7.4 Serial section 3D reconstructions of antennal sensilla Placodea of a parasitic wasp
Cotesia glomerata. Sample was high pressure frozen, epoxy freeze substituted, embedded and
observed at room temperature. a 12.5 9 13.0 9 0.7 lm3, 11 sections, section thickness 70 nm.
b 3D model of the chitin organization of Placodea and Coeloconica sensillas. Red arrows
indicate the protrusion cavity of both sensillas
section of the sample. If the section is of high quality, it may be collected and used
for correlative electron microscopy analysis. The possibility of acquiring a series
of consecutive AFM images of the same area of a sample surface after having
sectioned it with a certain fixed thickness facilitates 3D reconstructions of struc-
tural features within the sample volume. In this special design, sample and probe
remain motionless relatively to each other during the sectioning, which minimizes
spatial shift and potential misalignments between consecutive AFM images down
to a level of below 1 lm. The presented device can be employed for investigation
of thermotropic dynamic processes in soft materials in situ as it provides the
possibility of investigating samples at a wide range of temperatures (from 50 to -
120 C). Temperature variation is only limited by the ultramicrotomes
cryo-chamber which uses liquid nitrogen as a cooling agent.
First, suitable samples can be frozen in the cryo-chamber of the instrument if
the glass transition temperature (Tg) of the sample is below 0 C or if the sample
consists of several phases with rather weak phase adhesion. Alternatively, the
investigation can be performed stepwise at several different temperatures, starting
from temperatures of up to 50 C and continuously going down to -120 C (e.g.
for samples consisting of several polymer phases with different Tg or samples
showing temperature dependent dynamic structural changes). Hydrated materials
(such as nanoliquids and almost all biological objects) can be frozen by applying
high pressure and then be transferred to the SNOTRA using a cryo transfer system.
The resented approach makes it possible to obtain 3D reconstructions of the
sample volume both at room temperature and at cryo conditions using serial
section tomography. Figure 7.4 represents a three-dimensional ultrastructure of the
7.3 Cryo AFM Serial Section Tomography 91
Fig. 7.5 Serial section 3D reconstructions of PA6/SAN samples at cryo conditions. Block face
was cryo-sectioned and immediately scanned at -80 C. a 7.9 9 6.2 9 0.75 lm3,
b 2.0 9 2.0 9 0.75 lm3, six sections each, section thickness 125 nm
antennal sensillas Placodea and Coeloconica of the parasitic wasp Cotesia

glomerata. The chitin organization of the antenna clearly indicates that both
sensillas are protruding through the cuticle simultaneously towards the main neural
pathway of the wasp, which indicates a simultaneous processing of sensory
information detected by both sensillas. Interestingly, the single Coeloconica
sensillum is localized within a large segment of the antenna. This makes a
conventional optical search for further TEM analyses of this area extremely
complicated. The AFM imaging of the block face of the sample between
sectioning processes helps to identify the precise area of interest in a simple and
rapid way. This makes SNOTRA very convenient for the analysis of rarely
distributed phases/defects within the area of interest.
Figure 7.5 represent a 3D reconstruction of a polyamide 6 (PA 6) and styrene-
acrylonitrile (SAN) polymer blend obtained at cryo conditions (-80 C). PA 6 and
SAN are immiscible and thus compatibilization of these polymers is an important
topic of investigation for an optimum combination of the beneficial material
properties of both blends. Distribution and shape of the PA 6 domains are the main
structural parameters of compatibilization; furthermore there is the stability of the
interfacial area between the two polymers. Both of these parameters can be
investigated only at low temperatures due to very weak interfacial domain
connections between PA6 and SAN. Usually PA 6 domains are displaced from the
matrix during the section preparation at room temperature. Stabilization of the
sample through low temperature makes it possible to overcome this problem and to
obtain information about real distributions of the polymer phases in the sample
volume. Currently we are working with the frozen hydrated system in order to get
a 3D reconstruction models of some pharmaceutical systems as well as biological
tissues.
In conclusion, SNOTRAs ability to detect thermotropical changes of ultra-
structure in a wide range of temperatures (from 50 to -120 C) at a nanometer
resolution immediately after the sectioning procedure has been performed,
provides unique possibilities: firstly, the original native organization of soft
materials can be visualized and a clear understanding of artifacts and structural

changeswhich are usually introduced by conventional room temperature
microscopic techniquesis obtained. Likewise, the instrument allows the
performance of freeze fracturing analyses which provide additional information
about polymer packing density, morphology and orientation of chains without
contrast enhancing procedures such as conventional carbon/metal replica prepa-
ration. Finally, SNOTRA permits the visualization of samples immediately after
sectioning with a subsequent serial section 3D reconstruction of morphological
details at a wide range of temperatures. This paves the way for a new area of soft
material microscopy with an extremely bright future [31].
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Part III
Polymer Based Matter
Chapter 8
Morphology in OrganicInorganic
Composites
8.1 Introduction
Polymeric materials are generally reinforced with inorganic fillers in order to

reduce cost or to enhance mechanical, thermal, rheological and barrier properties.
The fillers used have different geometrical dimensions and thus affect the polymer
properties differently. For example, silica (and calcium carbonate) particles are
generally spherical (aspect ratio near to 1) and enhance the strength of the poly-
meric materials. On the other hand, clay and graphene particles are platy in nature
and enhance the mechanical, barrier and electrical properties of the polymers.
Fibers or nanotubes have the highest aspect ratio and enhance the longitudinal
strength as well as electrical properties of the polymer materials. Most common
factor affecting the filler performance is the dispersion and distribution of the filler
particles in the polymer matrix. Good dispersion and distribution of the filler
particles is mandatory to achieve efficient interface between the organic inorganic
components. Various microscopy techniques constitute most powerful methods to
characterize the morphology of the organicinorganic materials as described by
the diverse examples presented in the following sections. The characterization of
nanocomposites also poses additional challenges owing to the large number of
nano-sized particles. The microscopic characterization also acts as a quality
control tool as the poorly dispersed systems can be improved by changing the
process parameters. Apart from characterization of the distribution and dispersion
of filler particles, microscopy techniques also provide information on the align-
ment of the platy and fibrous particles.

98 8 Morphology in OrganicInorganic Composites
Fig. 8.1 SEM micrographs of PE/CaCO3 (irregular shaped) composites. a Good distribution and
b Poor distribution. The composites were plasma (oxygen) etched, platinum coated (3 nm) and
sectioned at cryo (-80 C) conditions
8.2 What is the Morphology of the Composite Material

Containing Low Aspect Ratio/Platy/Fibrous Filler
Particles?
Scanning electron micrographs (SEM) in Fig. 8.1 show the morphology of the
polyethylene nanocomposites with irregular (microparticles) shaped calcium car-
bonate particles. Figure 8.1a indicated a good distribution of the calcium carbonate
particles, however, some particles were removed during sectioning leaving behind
holes, which indicated that the adhesion between the polymer and inorganic
particles was poor. The size distribution of the particles themselves was also very
broad. Figure 8.1b shows the presence of agglomerates of calcium carbonate
particles when the weight fraction of inorganic particles was increased in the
composites. Pulling out of the particles from the matrix was similarly observed.
SEM micrographs in Fig. 8.2 depict the morphology of the composites when
spherical nanoparticles were used. The size distribution of the particles was also
more uniform as compared to Fig. 8.1. The distribution and dispersion of the
particles in polyethylene was much better as compared to microparticles. Pulling
of the particles during sectioning was also absent indicating better stability of the
particles in the composite structure. However, the sectioned surface of the polymer
was rough indicated that polymer was still flexible even at the cryo conditions used
for sectioning. Some agglomerates were observed as shown in Figs. 8.2a and c.
Figure 8.2b indicated a good distribution and dispersion of the filler particles
whereas varying degrees of filler dispersion were observed in Fig. 8.2c. Figure 8.3
shows the transmission electron micrographs (TEM) of silica-coated polymer
particles using surface functionalized polystyrene latex particles as well as bare
8.2 What is the Morphology of the Composite Material 99
Fig. 8.2 SEM micrographs of PE/CaCO3 (regular; spherical) nanocomposites. a Presence of

large agglomerates. b Good dispersion and distribution and c Mixed morphology with varying
degrees of filler dispersion. Nanocomposites were plasma (oxygen) etched, platinum coated
(3 nm) and sectioned at cryo (-80 C) conditions
polystyrene particles [1]. The morphology of the resulting organicinorganic

hybrid was significantly different. Using functionalized polystyrene particles led to
the generation of a uniform layer of silica particles around them. In the case of
non-functionalized polystyrene particles, the silica adsorption was non-uniform.
Apart from that, a fraction of silica particles was also free in the dispersion
medium. Figure 8.4 shows the atomic force (AFM) phase images of the silica/
rubber nanocomposite [2] at different magnifications. Dark regions in the micro-
graphs correspond to silica filler particles. Though the silica particles were dis-
tributed throughout the rubber matrix, the dispersion of silica particles was
however not uniform as agglomerates of silica particles were present in the
polymer matrix. The agglomerates themselves were also of different sizes.
Fig. 8.3 TEM micrographs of silica-coated polymer particles using a Surface functionalized
polystyrene particles and b Bare polystyrene particles. Scale bar reads 100 nm. Reprinted from
reference 1 with permission (American Chemical Society)
Fig. 8.4 AFM phase images of the silica/rubber nanocomposite. The block face has been
prepared using cryo ultramicrotomy. Dark regions correspond to silica filler particles. Scale bars:
1 lm in a and 300 nm in b. Reproduced from reference 2 (Nova Science Publishers)
Figure 8.5 shows the SEM micrographs of good and poor dispersion of
montmorillonite clay platelets in polypropylene [3]. Nanocomposite in Fig. 8.5a
was plasma (oxygen) etched, platinum coated (3 nm) and sectioned at
Fig. 8.5 SEM micrographs of polypropylene clay nanocomposites. a Poor filler dispersion and
b good dispersion. Nanocomposite in Fig. 8.5a was plasma (oxygen) etched, platinum coated
(3 nm) and sectioned at cryo (-80 C) conditions. Material in Fig. 8.5b represents the fracture
surface of the nanocomposite material after freezing in liquid nitrogen. Figure 8.5b is reproduced
from reference 3 with permission (Sage Publishers)
Fig. 8.6 TEM micrographs of polyurethane clay nanocomposites. a Exfoliated morphology and
b intercalated morphology. Reproduced from reference 4 with permission (American Chemical
Society)
cryo (-80 C) conditions, whereas material in Fig. 8.5b represents the fracture
surface of the nanocomposite material after freezing in liquid nitrogen. The
fracture surface indicated a random alignment of the filler platelets. The stacked
platelets forming agglomerate in Fig. 8.5a indicated that the polymer chains could
not intercalate these stacks owing to either polarity mismatch between the filler
Fig. 8.7 TEM micrographs of polymer clay nanocomposites depicting various states of filler
dispersion in the polymer matrix. a Exfoliated. b Intercalated and c un-intercalated. Reproduced
from reference 5 with permission (WileyVCH)
and the polymer chains or due to insufficient shear forces in the high temperature
melt mixer. It should be noted that the stacked aluminosilicate platelets in the
structure of the clay filler are better characterized by TEM rather than SEM, as
individual platelets can be detected. Figure 8.6 shows the TEM micrographs of
polyurethane clay nanocomposites [4]. In Fig. 8.6a, filler stacks were exfoliated to
single platelets in the polymer leading to exfoliated morphology. On the other
hand, in Fig. 8.6b, the stacks were reduced in thickness as well as interlayers
between the platelets in the stack were intercalated by the polymer chains leading
to intercalated morphology. Figure 8.7 similarly shows three different dispersion
states of montmorillonite filler in the polymer [5]. Figure 8.7a represents an
exfoliated morphology where the filler platelets have been delaminated as single
layers. Figure 8.7b is the intercalated morphology whereas Fig. 8.7c describes
Fig. 8.8 TEM micrographs of thermoplastic poly(urethane) nanocomposites matrix filled with
a unexfoliated graphite and b TEGO, processed by melt mixing. Micrographs c and d show
TEGO/polyurethane composites produced by solution blending and in situ polymerization
showing further exfoliation. Reproduced from reference 6 and 7 with permission (ACS and
Elsevier)
un-intercalated morphology where the stacks of the filler remained unchanged

after adding to the polymer matrix. Such a composite is also called a macro-
composite. In general, a combination of these three morphologies is more likely to
be present in the composites as compared to any single idealized morphological
state. Figures 8.8 and 8.9 depict the morphological characterization of nanocom-
posites with graphite oxide as filler. In Fig. 8.8a and b, TEM micrographs of
thermoplastic poly(urethane) nanocomposites matrix filled with unexfoliated
graphite and thermally expanded graphite oxide (TEGO) are shown [6, 7]. These
composites were processed by melt mixing and better dispersion of the TEGO
filler was clearly visible. When the composites were synthesized by solution
blending and in situ polymerization as shown in micrographs (c) and (d) respec-
tively, the state of filler dispersion was improved further. Thus, it indicated that the
synthetic methods can also be fine-tuned based on the information generated from
microscopic evaluation of the composite morphology. Further examples of the
Fig. 8.9 TEM images of SAN and PC nanocomposites containing 7.5 wt% of TrGO.
Reproduced from reference 8 with permission (Wiley Interscience)
dispersion of graphite oxide in the polymer matrices are depicted in Fig. 8.9 [8].
TEM images of styrene-co-acrylonitrile (SAN) and polycarbonate (PC) nano-
composites containing 7.5 wt% of thermally reduced graphite oxide (TrGO) at two
different magnifications have been demonstrated.
The morphological characterization of the composites with fibrous fillers is
demonstrated in Figs. 8.10 and 8.11. In Fig. 8.10a, SEM image of the fracture
surface for 5 wt% multiwall carbon nanotubes (MWCNT) composite with poly-
carbonate is shown [9]. Though the nanotubes are observed to be uniformly dis-
persed in the polymer matrix, varying extents of pulling of nanotubes from the
matrix were observed signifying weak polymer filler interface. Figure 8.10b
describes TEM image of a poly(vinyl alcohol) (PVA)-MWCNT composite film
[10]. The nanotubes are observed to have distribution in their length. Figure 8.11
also shows the SEM micrographs of poly(methyl methacrylate) (PMMA)-carbon
Fig. 8.10 a SEM image of

the fracture surface for 5 wt%
MWCNT in polycarbonate.
Reproduced from reference 9
with permission (Elsevier).
b TEM image of a poly(vinyl
alcohol) (PVA)-MWCNT
composite film. Reproduced
from reference 10 with
permission (ACS)
nanotube composites in the fractured area after tensile test for treated and
untreated nanotubes [11]. The treated nanotubes were observed to be covered with
polymer leading to better interaction between the two.Interactions between two
different types of inorganic materials can also be studied microscopically. Fig-
ure 8.12 shows the TEM images of gold nanoparticles attached to poly(methac-
ryloyl b-alanine) (PMBA)-coated MWCNT at two different magnifications [12].
The immobilization of the nanoparticles on the surface of the nanotubes is clearly
visible and only negligible amount of free nanoparticles is observed.
8.3 Do the Filler Substrates with Different Charge Densities

Affect the Morphology?
In the case of platy fillers like alumino-silicates, one important parameter defining
the nature of the substrate surface is the amount of charges present per unit area
(also defined as cation exchange capacity). Thus, it is also important to
Fig. 8.11 SEM micrographs of poly(methyl methacrylate) (PMMA)-carbon nanotube compos-

ites after tensile test. a Treated-MWCNT composite and b untreated-MWCNT. Reproduced from
reference 11 with permission (ACS)
characterize if such differences in the number of layer charges also affect the
morphology of the composite. Figure 8.13a and b shows the TEM images of the
epoxy nanocomposites with lower layer charge density montmorillonite and higher
layer charge density vermiculite minerals respectively. The lower charge density
filler particles were observed to be extensively bent and folded, whereas such a
phenomenon was absent in the composite with higher layer charge density min-
eral. Similar behavior is also indicated by the high resolution images of the
composites as shown in Fig. 8.14. The images Fig. 8.14a and b representing
composite with lower charge density mineral depicted lower stack thickness, but
the stacks were flexible [13]. On the other hand, the image Fig. 8.14c describing
the composite with higher layer charge mineral indicated a higher stack thickness,
but stiff morphology.
8.4 Do Different Filler Surface Modifications Affect Morphology? 107
Fig. 8.12 TEM images of a gold nanoparticles attached to poly(methacryloyl b-alanine)

(PMBA)-coated MWCNT and b high magnification image of immobilization of gold particles on
nanotubes. Reproduced from reference 12 with permission (IOP Publishing)
8.4 Do Different Filler Surface Modifications Affect

Morphology?
Filler surfaces are generally treated with organic modifications in order to enhance
their compatibility with the polymer matrices. Different surface modifications
owing to their specific interactions with the polymer matrices can lead to different
morphologies in the composites. Such changes in morphology can be characterized
by microscopy as shown in Fig. 8.15 [4]. TEM micrographs of polyurethane
montmorillonite nanocomposites depicting the morphology evolution as a function
Fig. 8.13 Lower magnification TEM micrographs of epoxy nanocomposites with a montmoril-
lonite and b vermiculite as fillers indicating the effect of different substrate or cation exchange
capacity. The scales read 200 and 500 nm respectively
of filler surface modification are shown. The filler in case of Fig. 8.15a is treated
with a polar modification, which led to exfoliated morphology of the filler in the
composites. The filler in case of Fig. 8.15b has partially polar modification which
resulted in mixed exfoliated-intercalated morphology. Owing to the polarity
mismatch of the non-polar modification with polar polymer in Fig. 8.15c, only
intercalated morphology was observed.
8.5 What is the State of Filler Alignment in the Composite?
The alignment of the platy and fibrous fillers in the polymer matrix is also of
importance as it impacts the filler performance. As an example, the aligned
platelets are much better in reducing the gas permeation through the polymers as
compared to the misaligned ones. Characterization of state of filler alignment is a
strong characteristic of microscopy techniques as is evident from the Figs. 8.5, 8.6,
8.7, 8.8, 8.9, 8.10, 8.11, 8.12, 8.13, 8.14, 8.15. In some cases, the misalignment is
preferred in order to provide isotropic characteristics to the composites. On the
other hand, alignment is occasionally induced by stretching or shearing the
composites materials in a particular direction. Apart from alignment, bending and
folding of the flexible filler layers in the polymer matrices is also characterized by
microscopy as depicted specifically in Figs. 8.13 and 8.14.
8.6 How is the Morphology Developed 109
Fig. 8.14 Higher magnification TEM micrographs of epoxy nanocomposites with a, b montmo-
rillonite and c vermiculite as fillers indicating the effect of different substrate or cation exchange
capacity. The bending and folding of the montmorillonite layers is also visible, whereas such a
phenomenon is not present in case of vermiculite filler. Image 8.14a and b are reproduced from
reference 13 with permission (ACS)
8.6 How is the Morphology Developed When Polymer is

Dispersed in Polymer?
The examples demonstrated so far deal with the organicinorganic materials.

However, polymers are also dispersed in polymers to achieve specific advantages
(like impact strength) and it is equally important to characterize the morphology of
the dispersed polymer phase in the continuous matrix phase. Figure 8.16 shows the
morphology of the 85PP/15PA6 and 70PP/30PA6 blends [14]. The cryo-fractured
surfaces were shown before and after the extraction of the dispersed phase using
Fig. 8.15 TEM micrographs of polyurethane montmorillonite nanocomposites depicting the

morphology evolution as a function of filler surface modification. a Polar modification. b Partially
polar modification and c non-polar modification. Reproduced from reference 4 with permission
(ACS)
HCOOH. Even on increasing the amount of the dispersed phase from 15 to 30 %,

the dispersion was still uniform, though the dispersed phase had large distribution
in the size. Figure 8.17 shows the SEM microphotographs of the poly(e-capro-
lactone) (PCL)/chitosan blend fibers with different extents of constituent polymers
[15]. The micrographs were taken after extracting the PCL component of the
blend. Change in morphology of the fibers on changing the composition of the
blend is clearly visible.
8.7 What Additional Information Can Be Generated 111
Fig. 8.16 Morphology of the 85PP/15PA6 a, a0 and 70PP/30PA6 b, b0 blends. A and B are
cryofractured surface, whereas a0 and b0 are cryofractured and have PA6 phase extracted using
HCOOH. Reproduced from reference 14 with permission (Elsevier). PP: polypropylene, PA6:
polyamide 6
8.7 What Additional Information Can Be Generated About

Composites on Combining Different Techniques?
Figure 8.18 shows an example of synergistic combination of TEM and energy

dispersive X-ray analysis techniques. It reports TEM/EDX analysis of the rubber
nanocomposite which has been reinforced by calcium carbonate and silica parti-
cles [2]. Apart from the information on the state of dispersion of the filler, EDX
spectrums also determine which elements are present at the particular position of
the sample.
Fig. 8.17 SEM microphotographs of the poly(e-caprolactone) (PCL)/chitosan (CHT) blend

fibers with a 75 parts CHT. b 50 parts CHT and c 25 parts CHT after extracting the PCL
component of the blend. Reproduced from reference 15 with permission (Elsevier)
References 113
Fig. 8.18 TEM/EDX analysis of the rubber nanocomposite which has been reinforced with
calcium carbonate and silica particles. EDX spectrums determine which elements are present at
the particular position of the probe. Position (1) on TEM bright field micrograph represents EDX
spectrum 1, and position (2) spectrum 2. Ultrathin section of the rubber nanocomposite was
prepared at cryo conditions (-190 C) and then stained with OsO4. Reproduced from reference 2
(Nova Science Publishers)
References
1. Tissot, I., Novat, C., Lefebvre, F., Bourgeat-Lami, E.: Macromolecules 34, 5737 (2001)
2. Matsko, N.B. In: Mittal, V. (ed.) Advances in Polymer Nanocomposites Technology, p. 407.
Nova Science Publishers, New York (2010)
3. Mittal, V.: J. Thermoplast. Compos. Mater. 20, 575599 (2007)
4. Osman, M.A., Mittal, V., Morbidelli, M., Suter, U.W.: Macromolecules 36, 98519858
(2003)
5. Mittal, V. In: Mittal, V. (ed.) Optimization of Polymer Nanocomposite Properties, p. 1. Wiley
VCH Verlag, Weinheim (2010)
6. Kim, H., Miura, Y., Macosko, C.W.: Chem. Mater. 22, 34413450 (2010)
7. Potts, J.R., Dreyer, D.R., Bielawski, C.W., Ruoff, R.S.: Polymer 52, 525 (2011)
8. Steurer, P., Wissert, R., Thomann, R., Mulhaupt, R.: Macromol. Rapid Comm 30, 31627
(2009)
9. Eitan, A., Fisher, F.T., Andrews, R., Brinson, L.C., Schadler, L.S.: Compos. Sci. Technol. 66,
11621173 (2006)
10. Lin, Y., Zhou, B., Fernando, K.A.S., Liu, P., Allard, L.F., Sun, Y.-P.: Macromolecules 36,
71997204 (2003)
11. Velasco-Santos, C., Martinez-Hernandez, A.L., Fisher, F.T., Ruoff, R., Castano, V.M.: Chem.
Mater. 15, 44704475 (2003)
12. Kumar, N.A., Bund, A., Cho, B.G., Lim, K.T., Jeong, Y.T.: Nanotechnology 20, 225608
(2009)
(2004)
14. Wang, D., Li, Y., Xie, X.-M., Guo, B.-H.: Polymer 52, 191200 (2011)
15. Malheiro, V.N., Caridade, S.G., Alves, N.M., Mano, J.F.: Acta Biomater. 6, 418428 (2010)
Chapter 9
Interface Morphology
9.1 Introduction
In principle, this chapter is the continuation of the previous chapter where the
dispersion of various types of fillers in polymer matrices was discussed. However,
in this chapter, more examples pertaining to the interfacial morphology evolution
in organicinorganic composites as well as polymer blends are demonstrated. For
example, in the case of nanocomposites with multiple components, specific
interactions of some components with filler phase can lead to slightly different
morphology at the filler matrix interface as compared to the bulk. Similarly, the
interface is very dynamic in the case of multi-component blend systems and is
significantly affected even when slight changes in the blend composition are made.
The following sections demonstrate the characterization of these aspects associ-
ated with the interface evolution.
9.2 What is the Impact of Small Amount of Compatibilizer

on the Polymer Morphology?
Compatibilizers are generally added to the polymers in order to generate

compatibility between the two phases of the composites. For example, in order
to compatibilize a polar filler surface with non-polar polymer, an amphiphilic
compatibilizer (with both polar and non-polar components) can be added which
leads to physical binding of the filler particles as well as matrix polymer
chains. Compatibilizers, however, can impact the microstructure (e.g., crystal-
linity) and mechanical performance of the polymers significantly, thus,
requiring an accurate characterization of their influence. Figures 9.1 and 9.2
show the AFM and TEM images of a high molecular weight polyethylene
116 9 Interface Morphology
Fig. 9.1 AFM images of the

polyethylene mixed with
a crystalline compatibilizer
and b amorphous
compatibilizer. The
compatibilizer is chlorinated
polyethylene with lower
molecular weight
polymer mixed with a crystalline as well as an amorphous compatibilizer. The

compatibilizer used in this case is chlorinated polyethylene with lower
molecular weight. The amount of compatibilizer was 5 % by weight. As is
visible in these images, the addition of a small amount of crystalline com-
patibilizer to semi-crystalline polymer retained its crystalline nature (Figs. 9.1a
9.2 What is the Impact of Small Amount of Compatibilizer 117
Fig. 9.2 TEM images of the

polyethylene mixed with
a crystalline compatibilizer
and b amorphous
compatibilizer. The
compatibilizer is chlorinated
polyethylene with lower
molecular weight
and 9.2a), whereas the crystallinity was significantly disturbed on addition of

amorphous compatibilizer to the polymer (Figs. 9.1b and 9.2b). Thus, the
morphology of the polymer can be affected by different additives added to the
polymers for processing or stability, even if their amount is quite low.
Fig. 9.3 AFM image of the

polyethylene/compatibilizer/
graphene nanocomposite
indicating the morphology of
the interface between the
polymer and the filler
Fig. 9.4 EDX analysis of the polyethylene/compatibilizer/graphene nanocomposite to evaluate

the morphology of the interface between the polymer and the filler
9.3 What is the Composition of Interface in Polymer/Compatibilizer/Filler 119
Fig. 9.5 ac TEM micrographs of a blend of EA, PA and MWNTs at a 2 wt% MWNTs, 10 min
mixing; b 2 wt% MWNTs, 60 min mixing and c 0.5 wt% MWNTs, 10 min mixing; and d SEM
micrograph of a cryofractured blend of EA, PA and 2 wt% MWNTs, 10 min mixing. Reproduced
from Ref. [1] with permission (Elsevier)
9.3 What is the Composition of Interface in Polymer/

Compatibilizer/Filler Nanocomposite?
As mentioned above, the specific interactions of a certain component in the com-

posites with the filler phase can lead to a different interfacial morphology as com-
pared to bulk material. Owing to the amphiphilic nature of the compatibilizers added
Fig. 9.6 a SEM micrograph

of a PVC/PS blend containing
20 % PS and b SEM
micrograph of PMMA/PS
blend containing 40 % PS.
Reproduced from Ref. [2]
with permission (Elsevier)
to the polymers, the polar part of such compatibilizer may specifically interact with
the polar surface of the filler thus leading to higher concentration of compatibilizer at
the interface. Figures 9.3 and 9.4 show the characterization of such interfacial
morphology for polyethylene graphene nanocomposites compatibilized with chlo-
rinated polyethylene. In the AFM image shown in Fig. 9.3, the compatibilizer (seen
as whitish areas in the micrograph) is observed to be distributed uniformly through
the matrix. However, the concentration of compatibilizer seemed to be higher at the
interface with the graphene particles (as indicated by thick whitish band along the
length of graphene platelets in the middle of the image). This was further confirmed
by energy dispersive X-ray analysis of the system presented in Fig. 9.4. On the
surface of graphene, presence of chlorine was detected, confirming the AFM finding
of higher concentration of chlorinated polyethylene near to graphene owing to polar
interactions between the two. EDX analysis could not detect chlorinated polyeth-
ylene in the matrix probably owing to lower concentration.
9.3 What is the Composition of Interface in Polymer/Compatibilizer/Filler 121
Fig. 9.7 SEM micrographs of HDPE/PS/PMMA/PVDF blends after microtoming and extraction
of the PMMA phase; a 10/15/15/60, b 20/15/15/50, c 30/15/15/40, d 40/15/15/30, e 50/15/15/20
and f 60/15/15/10. Reproduced from Ref. [3] with permission (Elsevier)
9.4 What are the Morphological Features of Polymer Blends

Stabilized by Nanoparticles?
Inorganic nanoparticles are also used to compatibilize the polymer blends. How-
ever, the generated morphology is dependent significantly on the blend compo-
sitions, amount of inorganic particles as well as processing conditions. Figure 9.5
Fig. 9.8 FIB-AFM images of PS/PP/HDPE 10/45/45 vol% blends after 30 min of quiescent
annealing with a 0 % copolymer; b 1 % SEB1 and c 1 % SEB3. Reproduced from Ref [4] with
permission (Elsevier)
shows TEM micrographs of a blend of ethylenemethyl acrylate random

copolymer (EA) and polyamide (PA), which was stabilized with multi-walled
carbon nanotubes (MWNTs) [1]. Various processing conditions like (a) 2 wt%
MWNTs, 10 min mixing, (b) 2 wt% MWNTs, 60 min mixing and (c) 0.5 wt%
MWNTs, 10 min mixing were used. Figure 9.5d also describes a SEM micrograph
of a cryofractured blend of EA, PA and 2 wt% MWNTs, 10 min mixing. In this
case, no inclusions of EA phase in the PA phase were observed even after 60 min
of mixing. The MWNTs are uniformly localized at the interface, as also confirmed
by the SEM analysis.
9.4 What are the Morphological Features of Polymer Blends 123
Fig. 9.9 SEM micrographs of a inner layer, b core region, and c outer layer at two positions in
water assisted injection molded (WAIM) PP/PA6 curved duct. Water pressure: 10 MPa; melt
temperature: 230 C; injection speed: 50%. Reproduced from Ref. [5] with permission (Elsevier)
Fig. 9.10 3D model of

acrylonitrilebutadiene
styrene/polyamide 6 polymer
blend structure
(8.75 9 5.0 9 1.0 lm,
25 sections, spaces between
Sections. 40 nm).
with permission (Nova
Science Publishers)
Fig. 9.11 AFM a height and b phase images of a block copolymer material. Reproduced from
Ref. [6] with permission (Nova Science Publishers)
9.5 How Does the Interfacial Morphology in Blends Change

as a Function of Different Compositions?
As mentioned above, the morphology of the blends (especially multi-component

systems) is affected significantly by changes in the composition. For example,
Fig. 9.6 shows SEM micrographs of a PVC/PS blend containing 20 % PS and
PMMA/PS blend containing 40 % PS [2]. The completely different morphologies
in these blend systems are clearly evident. Similarly, Fig. 9.7 depicts SEM
micrographs of HDPE/PS/PMMA/PVDF blends after microtoming and extraction
of the PMMA phase using compositions of four components as a) 10/15/15/60, b)
20/15/15/50, c) 30/15/15/40, d) 40/15/15/30, e) 50/15/15/20 and f) 60/15/15/10 [3].
As PMMA is extracted, it is present as voids separating the continuous parts of the
PS layer attached to HDPE and continuous PVDF. As is evident that at low
concentration of HDPE in the range of 10 % and 20 %, droplets of HDPE are
encapsulated by the PS phase, and both are encapsulated by PMMA in a matrix of
PVDF. The morphology was opposite when PVDF concentration was as low as
10 % and 20 %, where PVDF droplets are encapsulated by PMMA phase, and
both are encapsulated by PS within an HDPE matrix. Similarly, other morpho-
logical differences are observed on increasing or decreasing the concentration of
different components. Another example is shown in Fig. 9.8, which shows the
focused ion beam (FIB)-AFM images of PS/PP/HDPE 10/45/45 vol% blends after
30 min of quiescent annealing with a) 0 % copolymer; b) 1 % SEB1 and c) 1 %
SEB3 [4]. SEB copolymer corresponds to styrene(ethylenebutylene) diblock
polymers and SEB3 has higher percentage of styrene than SEB1.
9.6 How Does the Morphology in the Blends Evolve 125
9.6 How Does the Morphology in the Blends Evolve When

Specific Processing Conditions are Used?
Figure 9.9 shows the SEM micrographs of (a) inner layer, (b) core region, and (c)
outer layer at two positions in water assisted injection molded (WAIM) PP (larger
component)/PA6 (smaller component) curved duct [5]. Water pressure of 10 MPa,
melt temperature of 230 C and injection speed of 50 % was used for the process.
It is evident that morphology of the dispersed phase changes in three different
regions, i.e., inner layer, core region and outer layer. On the whole, the dispersed
phase exhibits a larger degree deformation in all three regions of position 2 than
that in corresponding regions of position 1.
9.7 How Does the Morphology in the Blends Seem in 3D

Microscopic Model?
Figure 9.10 depicts a 3D model of acrylonitrilebutadienestyrene (ABS)/poly-

amide 6 (PA6) polymer blend structure (8.75 9 5.0 9 1.0 lm, 25 sections, spaces
between Sections. 40 nm) [6]. The serial section tomography has been achieved
by a specially designed AFM/UC6NT ultramicrotome at room temperature. No
special sample preparation was performed of ABS/PA6 samples besides regular
ultramicrotome trimming. The 3D volume image reveals submicron spherical
clusters of ABS in PA6 matrix.
9.8 What is the Morphology in a Block Copolymer?
The earlier examples characterized the morphology of the polymer blends systems,
in which the polymers were mixed physically. Figure 9.11 shows the AFM char-
acterization of a block copolymer material, in which the two polymer blocks are
chemically bound to each other [6]. The soft and hard phases are clearly visible.
References
1. Baudouin, A.-C., Auhl, D., Tao, F.F., Devaux, J., Bailly, C.: Polymer 52, 149156 (2011)
2. Fekete, E., Foldes, E., Pukanszky, B.: Eur. Polym. J. 41, 727736 (2005)
3. Ravati, S., Favis, B.D.: Polymer 51, 36693684 (2010)
4. Virgilio, N., Desjardins, P., LEsperance, G., Favis, B.D.: Polymer 51, 14721484 (2010)
5. Huang, H.-X., Zhou, R.-H.: Polym. Test 29, 235244 (2010)
6. Matsko, N.B.: In: Mittal, V. (ed.) Advances in Polymer Nanocomposites Technology, p. 407.
Nova Science Publishers, New York (2010)
Chapter 10
Surface and Volume Characterization
10.1 Introduction
Both surface and volume morphology of the systems is required to be character-

ized as the resulting surface and bulk properties of the materials drive their
applications. The control on the morphology and its tuning according to the
requirement is another characteristic which is generally optimized by microscopic
characterizations. These analyses can lead to vital information like surface
smoothness/hardness (which in turn affects the wetting and adsorption character-
istics of the surface), surface morphology (like strawberry, moon crater, hemi-
spherical morphology etc.), particle size and its distribution, porosity of the
particles, interactions between the components, defects present in the bulk of the
sample, overall stability/dispersion of the filler phase in the polymer matrix,
structure of the monoliths etc. The following sections demonstrate these analyses
for a wide range of systems. Apart from organic and organicinorganic systems, a
brief discussion on the surface and volume characterization of inorganic particles
has also been presented.
10.2 What is the Overall Morphology in the Volume

of the Sample?
This section is similar to characterization of the dispersion of the filler phase

described in earlier chapters. The analysis here is broadened to obtain information
on the overall morphology in the volume of the sample by using lower magnifi-
cation images. Figure 10.1 shows the TEM micrographs of slurry of natural clays
Cloisite Na and Optigel CK in water. These clays slightly differ in their layer
charges. As their platelets are polar in nature, they exfoliate easily in water leading
128 10 Surface and Volume Characterization
Fig. 10.1 TEM micrographs

of slurry of natural clays
a Cloisite Na b Optigel CK in
water
to exfoliated morphology as observed in the micrographs. Only occasionally, the

presence of un-exfoliated clay particles is observed and overall morphology in the
volume of the samples is uniform. Figure 10.2 shows the TEM micrographs of
epoxy embedded benzyl (2-hydroxyethyl)methyloctadecylammonium (BzC18OH)
modified montmorillonite and vermiculite alunimo-silicates. The dark lines
10.2 What is the Overall Morphology in the Volume of the Sample? 129

of epoxy embedded benzyl
(2-hydroxyethyl)
methyloctadecylammonium
(BzC18OH) modified
a montmorillonite and
b vermiculite platelets. The
dark lines represent the cross-
section of the platelet layers
represent the cross-section of the alumino-silicate layers. As montmorillonite and

vermiculite vary significantly in their layer charge densities, significant differences
in the morphology of the filler platelets are generally observed. The montmoril-
lonite particles are observed to be distributed randomly and extensive bending and
folding is visible. On the other hand, vermiculite platelets are observed to be more
Fig. 10.3 Low magnification

images of a epoxy and b PU
nanocomposites with surface
modified montmorillonite
depicting the volume of the
nanocomposite materials
aligned and less flexible. The thickness of the particles is larger in the case of
vermiculite mineral. Figure 10.3 depicts the volume of the epoxy and PU nano-
composites with surface modified montmorillonite through their low magnification
images. Similar observations of misalignment, bending, folding of platelets can be
made. Similarly, TEM micrographs of epoxy graphene nanocomposite at 1.5 wt%
10.2 What is the Overall Morphology in the Volume of the Sample? 131

of epoxy graphene
nanocomposite at 1.5 wt%
filler. Reproduced from Ref.
[1] with permission (Elsevier)
filler content are shown in Fig. 10.4 [1]. Figure 10.4a shows that single graphene
platelets or stacks of graphene with thicknesses of several nanometers were uni-
formly dispersed throughout the polymer matrix. Complete embedding of the
particles in the epoxy resin was observed indicating an intercalated structure.
Figure 10.4b represents the fracture surface of the composite which indicated less
brittle failure as compared to pure polymer. Figure 10.5 shows the TEM

depicting the volume of
cyanate ester-multi walled
carbon nanotubes composites
containing 1 wt% filler.
micrographs depicting the volume of cyanate ester-multi walled carbon nanotubes

composites containing 1 wt% filler [2]. It is observed that the nanotubes form
certain aggregations, leaving some areas of the polymer matrix unfilled. Similarly,
Fig. 10.6 shows the volume morphology of the poly (2-hydroxyethyl acrylate)
(PHEA)/silica nanocomposites at different mass fractions of silica [3].
10.3 Which Defects in the Volume of the Samples

can be Detected?
Characterization of the volume morphology also leads to detection of defects in

the sample, which sometimes may not be visible from the surface. Figure 10.7
demonstrates an example of such defects through TEM micrographs of polyure-
thane (PU) montmorillonite nanocomposites. The drying and curing of the sample
10.3 Which Defects in the Volume of the Samples can be Detected? 133
Fig. 10.6 AFM phase image of the PHEA/silica hybrid nanocomposites. Different mass
fractions of silica: a 5 wt%, b 10 wt%, c 15 wt%, d 20 wt%, e 25 wt%, f 30 wt% were used.
Reproduced from Ref. [3] with permission (Elsevier)
was performed at atmospheric pressure, which resulted in the bubbles or mac-

rovoids in the nanocomposite films. The humidity or water vapor present in the
atmosphere could react with the isocyanate molecules of the prepolymer during
Fig. 10.7 a and b TEM

micrographs depicting the
volume morphology of PU
montmorillonite
nanocomposites when drying
and curing was performed at
atmospheric pressure
the polymer curing at atmospheric pressure leading to carbon dioxide evolution.

Owing to increasing viscosity during the curing process, after certain time, the
polymer may not allow the carbon dioxide bubbles to escape anymore, thus,
embedding them in the matrix. TEM of the cross-section of complete thickness of
the PU nanocomposite film is also shown in Fig. 10.8. The filler concentration at
the bottom of the film is observed to be higher than the top of the film which
10.3 Which Defects in the Volume of the Samples can be Detected? 135
Fig. 10.8 TEM micrograph

of the cross-section showing
the thickness of the PU
montmorillonite
nanocomposite film. The
scale reads 2000 nm
signified that the filler is not properly swollen and sediments at the bottom of the
film during the curing process.
10.4 How Does the Surface Morphology Change

as a Function of Synthesis Equipment?
The surface morphology of the particles can also be affected by the reaction
apparatus as described in Fig. 10.9. In the reaction flask with the contents stirred
by a magnetic stirrer, the obtained surface morphology of the particles is signifi-
cantly rough, whereas for the same system, smooth particles are generated when
synthesis is carried out in automatic reactor with mechanical stirring. It is thus
confirmed that the stirring of the reaction medium can play a significant role in
developing the surface morphology of the particles. In the automated lab reactor,
efficient as well as more controlled stirring takes place which helps to smoothen
the surface of the particles.
10.5 How Does the Surface Morphology of Particles Change

as a Function of Reaction Conditions?
Surface morphology of the particles can also be affected by the reaction conditions.
Emulsion polymerization (using a surfactant) is commonly used technique to
synthesize polymer nanoparticles. In a special case of generation of polymer
Fig. 10.9 SEM images

showing the comparison of
the surface morphology of the
particles prepared in
a reaction flask and
b automated reactor
particles in surfactant free emulsion polymerization, the surface morphology is very

sensitive to minute changes in synthesis methodology. Figure 10.10 demonstrates
the surface morphology of the polystyrene particles functionalized with a thin layer
of copolymer (of styrene and a functional monomer) in surfactant free emulsion
polymerization conditions [4]. By changing synthetic conditions like composition
of monomers, addition of monomers separately or together, addition of monomers
10.5 How Does the Surface Morphology 137
Fig. 10.10 Surface morphology of the polystyrene particles functionalized with a thin layer of
copolymer (of styrene and a functional monomer) as a function of changes in synthetic
conditions. Reproduced from Ref. [4] with permission (Elsevier)
slowly or as a shot etc., large changes in the resulting surface morphology of the
particles are observed. Different morphologies like orange-peel, strawberry and
moon crater morphology etc. are observed. Figure 10.11 also shows the charac-
terization of the same system for secondary nucleation (generation of secondary
particles of different size) of the polymer particles during the functionalization of
polystyrene particles to form a thin layer of copolymer (of styrene and a functional
monomer). As a function of changes in synthetic conditions, the extent of the
secondary nucleation was also affected. Similarly, Fig. 10.12 shows the SEM
images of the cationic amino-containing N-isopropylacrylamide-styrene P(St-NI-
PAM-AEM) copolymer latex particles latexes as a function of polymerization
conditions [5]. Changes in the surface morphology are clearly evident. Figure 10.13
shows the SEM images of poly(styrene) nanoparticles obtained at different
Fig. 10.11 Characterization of the secondary nucleation of the polymer particles during the
surface functionalization of polystyrene particles to form a thin layer of copolymer (of styrene
and a functional monomer). Figures a and b represent one system, whereas figures c and
d represent another system. Reproduced from Ref. [4] with permission (Elsevier)
experimental conditions of (a) DMF/MeOH, 1.5 mg/ml, @ 25 C (b) DMF/MeOH,

1.5 mg/ml, @ 4 C and (c) DMF/MeOH, 3 mg/ml, @ 4 C [6]. The process is
based on the use of a physical barrier, specifically dialysis membrane or semiper-
meable membranes that allow the passive transport of solvents to slow down the
mixing of the polymer solution with a nonsolvent; the dialysis membrane contains
the solution of the polymer. Figure 10.14 also shows SEM photomicrographs of the
P(GMAEDMA)/PS coreshell particles, produced by seeded emulsion copoly-
merization of glycidyl methacrylate (GMA) and the crosslinker monomer ethylene
glycol dimethacrylate (EDMA) in the presence of the PS core microspheres [7].
Increasing volume ratios of [EDMA]/[GMA] of 0.01, 0.05 and 0.2 were used. It is
observed that the diameter and size distribution of the produced microparticles is
Fig. 10.12 Scanning electron micrographs of P(St-NIPAM-AEM) latexes as a function of

polymerization conditions. AEM: aminoethylmethacrylate hydrochloride. Reproduced from Ref.
[5] with permission (Springer)
Fig. 10.13 SEM images of

poly(styrene) nanoparticles
obtained at different
experimental conditions:
a DMF/MeOH, 1.5 mg/ml,
@ 25 C, b DMF/MeOH,
1.5 mg/ml, @ 4 C and
c DMF/MeOH, 3 mg/ml,
@ 4 C. Reproduced from
reference 6 with permission
(ACS)
not affected by changing the volume ratio, however, a change in the morphology is
evident. Increasing the volume ratio results in more distorted morphology and
rough surfaces probably due to the grafting of P(GMAEDMA) copolymer chains
on the surface of the PS microspheres.
Fig. 10.14 SEM images depicting the surface morphology of P(GMAEDMA)/PS coreshell
micrometer-sized particles formed by emulsion polymerization of GMA and EDMA in the
presence of the PS microspheres. Different volume ratios of [EDMA]/[GMA] as a 1, b 5 and c 20
are used. Reproduced from Ref. [7] with permission (Elsevier)
Fig. 10.15 a TEM image showing the morphology of iron oxide nanoparticles surrounded by
gold nanoparticles and b EDX spectra of gold and iron nanoparticles (marked by circles and a
cross, respectively, in the inset). Reproduced from Ref. [8] with permission (ACS)
Fig. 10.16 TEM images of porous hematitesilica composite capsules: a, b before and c, d after
the elimination of hematite. Reproduced from Ref. [9] with permission (Wiley)
10.6 What is the Surface and Volume Morphology in Particle

Decorated Particles and Hollow Inorganic Particles?
Particle decorated particles are synthesized by first physically adhering gold

nanoparticles around hexagonally ordered micelles of the PS-b-2PVP diblock
copolymer film [8]. Iron oxide nanoparticles are then synthesized chemically in the
core area of the ordered micelles. Figure 10.15 shows the TEM image of iron
oxide nanoparticles surrounded by gold nanoparticles. EDX spectra of gold and
iron nanoparticles are also shown to further confirm the formation of the structure.
Figure 10.16 demonstrates TEM images of hematitesilica composite capsules
before and after HCl treatment [9]. Successful formation of hematitesilica
composite capsules and the elimination of hematite from the capsules to yield
10.6 What is the Surface and Volume Morphology 143
Fig. 10.17 ad SEM micrographs with different magnifications and e XRD pattern of the Cu2O
powder. Reproduced from Ref. [10] with permission (Wiley)
porous silica structures is evident. Figure 10.17 shows SEM micrographs of the
Cu2O powder with different magnifications and along with the XRD pattern [10].
The high-magnification images of open structures also reveal the multilayered
architecture in the radial direction.
10.7 What is the Volume Morphology in a Polymer Monolith

Formed by Association of Primary Particles?
Polymer monoliths are formed by binding of the polymer particles in a porous

network. The free particles are first aggregated physically and then coated/grafted
with a thin layer of cross-linked polymer. Homogenous coating of the particles in
monolith is required to achieve stability as well as performance in the monoliths.
However, it is also important to control the extent of polymer grafting as it may
affect the monolith porosity. Figure 10.18 shows the SEM micrographs of the
volume morphology of polymer monolith generated by using different processing
conditions as well as by using different extents of polymer grafting.
Fig. 10.18 SEM micrographs of the volume morphology of polymer monolith generated by
using different extents of polymer grafting
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1. Martin-Gallego, M., Verdejo, R., Lopez-Manchado, M.A., Sangermano, M.: Polymer 52,
46644669 (2011)
2. Luo, Z.P., Koo, J.H.: Mater. Lett. 62, 34933496 (2008)
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27752783 (2007)
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27662771 (2007)
Chapter 11
Confirmation of Surface Reactions
11.1 Introduction
Chemical reactions are carried out on the surface of various substrates in order to
generate specific functionalities or to change the surface properties of these sub-
strates. For example, polymeric brushes are grafted on the surface of clay platelets
in order to synthesize exfoliated nanocomposites. Similarly, the brushes are also
grafted from the surface of polymer particles in order to generate surface prop-
erties directing their various applications. It is thus of requirement to confirm the
surface reaction. Microscopy can be useful in the characterization of such polymer
grafting reactions on the surface by three ways:
1. Characterization of the expected effect on morphology as a result of surface
reactions: For example, the effect of grafting reaction on the surface of the clay
particles is their exfoliation. Thus, studying the extent of exfoliation would
reveal the success of surface reaction.
2. Visualizing the polymer layer on the surface.
3. Quantification of the polymer layer by energy dispersive X-ray analysis
(EDAX) and electron energy loss spectroscopy (EELS).
11.2 Has the Reaction on Clay Surface Taken Place?
Figure 11.1 shows the TEM micrographs of the reacted clay with initial coverage
of initiator corresponding to 70 % of its capacity and the rest with meth-
yltrioctylammonium [1]. The reacted clay showed the presence of some exfoliated
single layers. Apart from that, the clay stacks were observed to be decreased in
thickness owing to the penetration of the polymer indicating that the reaction was
not limited to the outer surface of the initial thick stacks and the polymerization
148 11 Confirmation of Surface Reactions

of clay modified with a
monomer in an amount
corresponding to 70 % of the
cation exchange capacity and
methyltrioctylammonium in
an amount corresponding to
remaining 30 % of the
capacity after solution
polymerization at 60 C. The
scale bar reads 20 nm.
was also achieved inside the clay interlayers. However, complete exfoliation was
not achieved. Similarly, Fig. 11.2 show the TEM micrographs of polystyreneclay
nanocomposites using Na-MMT and Ca-MMT [2]. The clay was modified with a
monomer containing modification and subsequent co-polymerization of these
monomer molecules with externally added monomer was carried out. It is evident
that micron sized clay particles do not exist in the nanocomposite, and the filler is
11.2 Has the Reaction on Clay Surface Taken Place? 149

of polystyreneclay
nanocomposites using
a Na-MMT and b Ca-MMT.
dispersed as nanolayers in the polystyrene matrix. This also confirms that the
polymer could be grafted on the surface of the filler platelets.
11.3 How can the Visual Characterization Confirm

the Surface Reaction?
Figure 11.3 shows the SEM micrographs of the polystyrene droplet formation on
the surface of mica platelets as a function of different reaction conditions [3]. By
changing the process conditions, thin films of polymer could also be generated.
Similarly, Fig. 11.4 also shows the AFM micrographs of clay platelets, clay
platelets after the immobilization of initiator molecules and platelets after polymer

polystyrene droplet formation
on the surface of mica:
a polymerization at 120 C
for 1 h, b polymerization at
100 C for 20 min, followed
by heating for 1 h at 59 C.
The scale bars read 100 nm.
with permission (ACS)
brush grafting from clay surfaces [4]. The change in the morphology of the surface
after every reaction step is evident. The immobilization of organic cation carrying
the initiator molecules provided full coverage over the clay layers. The polymer
grafting exhibited spherical or globular domains, but some portions of the surface
are less covered (less uniform).
11.3 How can the Visual Characterization Confirm the Surface Reaction? 151
Fig. 11.4 AFM micrographs of a clay platelets, b initiator adsorbed clay platelets, and c after
polymer brush grafting from clay surfaces. Reproduced from Ref. [4] with permission (ACS)
Fig. 11.5 TEM micrographs of a un-grafted multi walled carbon nanotubes and b-f polymer
grafted nanotubes as a function of different reaction conditions. Reproduced from Ref. [5] with
permission (ACS)
Similar surface reaction characterization can also be performed for carbon

nanotube functionalization processes. Figure 11.5 shows the TEM micrographs of
un-grafted and polymer grafted multi walled carbon nanotubes as a function of
different reaction conditions [5]. Poly(methyl methacrylate) (PMMA) was grafted
from the surface by using atom transfer radical polymerization (ATRP).
Fig. 11.6 High-resolution TEM images of a PS-pristine-MWNT and b PS-acid boiled-MWNT,

respectively. The samples were prepared by dropping solutions of PS-pristine-MWNTs and PS-
acid boiled-MWNTs in THF onto holey carbon-coated copper grids and then drying under
vacuum. Reproduced from Ref. [6] with permission (Elsevier)
Fig. 11.7 TEM images of a-b carboxylic acid-functionalized MWNTs, and c-d 4,4-bis(4,4-
isopropylidene diphenoxy)-bis(phthalic anhydride) (BisADA)2,2-dichloro-4,4-biphenyldi-
amine (DCB) (BisADADCB) grafted MWNTs. Reproduced from Ref. [7] with permission (ACS)
The average thicknesses of the polymer layers are also mentioned on the images. It
indicated that the thickness of polymer layer can be controlled. An example of
comparison of non-covalent and covalent functionalization of nanotubes is
depicted in Fig. 11.6 [6]. High-resolution TEM images of PS-pristine-MWNT and
PS-acid boiled-MWNT are compared. As is seen from the figure, the surface of the
PS-pristine-MWNT is uniformly attached with an amorphous layer of the polymer
along its length. On the other hand, the PS-acid boiled-MWNT is only attached
with the polymer at various sites confirming better surface functionalization of
nanotubes using pristine nanotubes. Another example of carboxylic acid-func-
tionalized MWNTs, and 4,4-bis(4,4-isopropylidene diphenoxy)-bis(phthalic
anhydride) (BisADA)2,2-dichloro-4,4-biphenyldiamine (DCB) (BisADA-DCB)
grafted MWNTs is shown in Fig. 11.7 [7]. A thin layer of BisADA-DCB with a
thickness of 520 nm is observed.
Some other miscellaneous example depicting the confirmation of surface
reactions by visual monitoring of the polymer are described below. Figure 11.8
Fig. 11.8 Tapping mode AFM images of a PMMA brushes formed on silicon substrate and
b clean silicon surface without polymer brushes. Reproduced from Ref. [8] with permission
(Elsevier)
Fig. 11.9 AFM images of a E9D and c E12D ZnO films and poly(N-isopropylacrylamide)-
modified b E9D and d E12D substrates. Reproduced from Ref. [9] with permission (Elsevier)
shows the tapping mode AFM images of PMMA brushes formed on silicon sub-
strate in comparison with clean silicon surface without polymer brushes [8]. Small
domains are observed in three-dimensional AFM image of the grafted PMMA
which can be attributed to the aggregation of PMMA chains on the surface. Fig-
ure 11.9 demonstrates the AFM images of E9D and E12D ZnO films before and
after poly(N-isopropylacrylamide) grafting [9]. E9D and E12D ZnO films were
deposited by the electrochemical deposition method at 1.3 V for 90 s and 120 s.
Fig. 11.10 a Stimulus-responsive polymer brushes (SRPB) used as carriers for bimetallic AuPt
NPs and (b) transmission electron microscopy (TEM) image of Au/Pt composite particle.
Reproduced from Ref. [10] with permission (WileyVCH)
As is observed from the images, the deposition of the ZnO layer leads to undu-
lations on the oxide surface, which decreases as the deposition time reaches 120 s.
When the polymer is grafted, the roughness of the substrate decreases further thus
confirming the presence of polymer film on the substrates. Colloidal polystyrene
particles, functionalized with polyelectrolyte (PEL) brushes can provide powerful
Fig. 11.11 Tapping mode AFM images of homopolymer brushes on the silicon wafer generated
by reverse ATRP: a 2-D image and b 3-D image. Reproduced from Ref. [11] with permission
(Elsevier)
Fig. 11.12 AFM micrographs of silica particles adsorbed on top of a poly(methylmethacrylate-

b-glycidylmethacrylate) (p(MMA-b-GMA)) diblock-copolymer brush. Reproduced from Ref.
[12] with permission (Elsevier)
means to reduce coagulation of metallic nanoparticles in solution [10]. It thus

produces nearly monodisperse suspensions of metal nanoparticles. Figure 11.10
shows such stimulus-responsive polymer brushes (SRPB) used as carriers for
bimetallic AuPt NPs. TEM image of a colloidal PS particle with PEL chains
emanating from its surface is shown which can then be used to immobilize various
metal ions within the PEL layer. Figure 11.11 reveals the 2D and 3D tapping mode
AFM images of homopolymer brushes on the silicon wafer generated by reverse
Fig. 11.13 a SEM micrograph of polystyrne (PS) particles, b SEM micrograph of the PS
particles functionalized on the surface with a functional monomer and c TEM of the polymer
brushes grafted from the surface of particles of Fig. 11.13b. Reproduced from Ref. [13, 14] with
ATRP [11]. The images demonstrate a smooth and uniform polymer brushes
surface with small thickness variation. Even the interactions between two com-
ponents can be microscopically detected. Figure 11.12 depicts AFM micrographs
of silica particles adsorbed on top of a poly(methylmethacrylate-b-glycidylmeth-
acrylate) (p(MMA-b-GMA)) diblock-copolymer brush [12]. Two different types of
dispersion states of silica particles on the surface can be detected.
Reactions on the surface of the polymer particles can also be similarly char-
acterized. Figure 11.13 shows the SEM micrograph of polystyrene (PS) particles,
PS particles functionalized on the surface with a functional monomer and poly(N-
isopropylacrylamide) brushes grafted from the surface of functionalized particles
[13, 14]. ATRP was used for the grafting reactions. The resultant morphologies in
three cases are completely different from each other. The homopolymer particles
Fig. 11.14 EELS performed on the particle shown in Fig. 11.13c indicating the distribution
profiles of a C, b O, c N and d W. Reproduced from Ref. [13] with permission (Elsevier)
are smooth on the surface, whereas functionalization with a functional monomer

results in orange-peel morphology. The grating of the brushes leads to a uniform
formation of a thick shell of the polymer brush confirming the success of the
process.
11.4 How can EDAX and EELS Quantify the Success

of Surface Reactions?
The surface reactions can also be confirmed by detecting the elements of the
polymer grafted or polymerized on the surface. It is required that these elements
are present only in the grafted content and not in the substrate. EDAX as well as
11.4 How can EDAX and EELS Quantify the Success of Surface Reactions? 159
Fig. 11.15 a Bright field TEM; b and c EELS spectra of the grafted polymer layer around the
particles of part (a)
EELS are most promising techniques to achieve these analyses. For example,
Fig. 11.14 show the EELS analysis of the particles of Fig. 11.13c [13]. As
carbon is present in substrate, grafted polymer as well as grid, a homogenous
carbon distribution signal was observed. The particle core remains black due to
its higher thickness. The oxygen and nitrogen elements of the grafted poly(N-
isopropylacrylamide) layer have distribution concentrated around the particle
confirming the presence of the grafted polymer layer. The analysis is also shown
for an element which is not present in the substrate or grafted content in order to
confirm that the results for C, O and N are not casual. Figure 11.15 also shows
the quantitative EELS analysis of the same system. It should be noted that C and
O signals are also contributed to by uranyl acetate used to stain the particles.
Signal of K also corresponded to the free radical initiator (potassium persulfate)
used during the emulsion polymerization reactions. As the negatively charged
sulfate ions initiate the polymer chains and in the process are present on the
surface of the particles owing to their hyrophilicity, the potassium ions are
present along them as counter ions. Figure 11.16 further demonstrates the rela-
tive thickness map of the grafted particles (for the particles in the bright field
TEM image of Fig. 11.16a) as analyzed with analytical TEM after staining. As
expected, the thickness decreased radially from the centre to the periphery of the
particles. The grafted brushes around the particles formed the thinnest region and
the particles were observed to be uniformly surrounded by such thin layer
(yellow in color). Particles shown in Fig. 11.16a are also analyzed with energy
filtered TEM to generate distribution patterns of N, Br and Cu atoms on and
around the particles as shown in Fig. 11.17. The N atom, the constituent of
grafted chains, was present not only around the particles, but in the background
also. This confirmed the presence of grafted chains around the particles as well
Fig. 11.16 a Bright field

TEM and b corresponding
relative thickness map of the
polymer grafted particles as
analyzed with analytical
TEM
as to a small extent free from the particles surface. On the other hand, the signal
corresponding to Br atoms, which form the ATRP initiator bound to the surface
of the particles, was strictly present on and around the particles, but not in the
background. Presence of Cu atom, which together with ligand forms ATRP
catalyst complex, around the surface of the particles also indicated certain
complexation with the initiator molecules.
References 161
Fig. 11.17 N, Br and Cu distribution in the polymer grafted particles as analyzed with energy
filtered TEM (EFTEM)
References
1. Mittal, V.: J Colloid Interface Sci 314, 141151 (2007)

2. Fu, X., Qutubuddin, S.: Polymer 42, 807813 (2001)
3. Velten, U., Tossati, S., Shelden, R.A., Caseri, W.R., Suter, U.W., Hermann, R., Muller, M.:
Langmuir 15, 69406945 (1999)
4. Fan, X., Xia, C., Fulghum, T., Park, M.-K., Locklin, J., Advincula, R.C.: Langmuir 19,
916923 (2003)
5. Kong, H., Gao, C., Yan, D.: J Am Chem Soc 126, 412413 (2004)
6. Liu, Y.-T., Zhao, W., Huang, Z.-Y., Gao, Y.-F., Xie, X.-M., Wang, X.-H., Ye, X.-Y.: Carbon
44, 16131616 (2006)
7. Ge, J.J., Zhang, D., Li, Q., Hou, H., Graham, M.J., Dai, L., Harris, F.W., Cheng, S.Z.D.: J Am
Chem Soc 127, 99849985 (2005)
8. Hou, S., Li, Z., Li, Q., Liu, Z.F.: Appl Surf Sci 222, 338345 (2004)
9. Chang, C.-J., Kuo, E.-H.: Thin Solid Films 519, 17551760 (2010)
10. Schrinner, M., Proch, S., Mei, Y., Kempe, R., Miyajima, N., Ballauff, M.: Adv Mater 20,
19281933 (2008)
11. Wang, Y.-P., Pei, X.-W., He, X.-Y., Lei, Z.-Q.: Eur Polym J 41, 737741 (2005)
12. Santer, S., Ruhe, J.: Polymer 45, 82798297 (2004)
13. Mittal, V., Matsko, N.B., Butte, A., Morbidelli, M.: Eur Polym J 43, 48684881 (2007)
14. Mittal, V., Matsko, N.B., Butte, A., Morbidelli, M.: Polymer 48, 28062817 (2007)
Chapter 12
Interactions Between Components
12.1 Introduction
Interactions can be of different kinds, e.g. interaction of polymer chains with

the surface of the filler, interactions of external species with the surface
(e.g. adsorption on the surface), interactions of particles surface with stimulants
like temperature, sonication, salt, solvent etc., or interactions between two
inorganic species etc. (also termed as decoration of one inorganic surface with the
particles of other). Such information can help to predict the behavior of the
materials, their aggregation tendency etc. The characterizations of such interac-
tions can be achieved either by analyzing the effect of such expected interactions
on the morphology or by actual visualization of the morphology of the compo-
nents. Though such evaluations generate information on the interaction between
the components, however, the nature of interaction (chemical or physical) is not
possible to be obtained. Various systems dealing with above mentioned interac-
tions between the different components and substrates are described in the
following sections.
12.2 Is There an Interaction Between the Polymer Chains

and the Filler Surface?
The filler surface in the case of polymer nanocomposites is organically modified in

order to enhance compatibility with the polymer chains. In case, the resulting
interactions between the polymer chains and filler surface are positive, the filler
particles exfoliate in the matrix. Thus, by studying the state of filler exfoliation
in polymer, it is possible to comment on the interactions between the two.
Figure 12.1 depicts TEM micrographs of epoxy montmorillonite nanocomposites,
164 12 Interactions Between Components

of epoxy montmorillonite
nanocomposites, containing
fillers modified with
a benzyldibutyl(2-
hydroxyethyl)ammonium
(Bz1OH), and b benzyl-
hexadecylammonium
(BzC16). Reproduced from
[1] with permission (ACS)
12.2 Is There an Interaction Between the Polymer Chains and the Filler Surface? 165
Fig. 12.2 TEM and EDX images of polyethylene graphene nanocomposite compatibilized with
chlorinated polyethylene
Fig. 12.3 TEM micrographs showing a adsorption and b desorption experiments of tobacco
mosaic virus on the polymer particles. Reproduced from [2] with permission (Elsevier)
Fig. 12.4 Adsorption of

negatively charged particles
on a negatively charged
sapphire disk and b positively
charged sapphire disk
Fig. 12.5 Optical

micrographs of polymer
particles grafted with
thermally responsive poly(N-
isopropylacrylamide)
polymer above and below
lower critical solution
temperature of 32 C:
a 20 C and b 40 C.
Reproduced from [3] with
permission (ACS)
Fig. 12.6 SEM micrographs

of polystyrene particles after
swelling with monomers
followed by salt
destabilization and
polymerization when the
polystyrene particles were
synthesized a without
surfactant and b with
surfactant
containing fillers modified with two different modifications benzyldibutyl(2-

hydroxyethyl)ammonium (Bz1OH) and benzylhexadecylammonium (BzC16) [1].
When polar modification of Bz1OH was used, the filler surface has a very good
interaction with the polar polymer leading to extensive filler exfoliation. On the
other hand, in the case of less polar modification BzC16, the filler platelets are
only intercalated with polymer signifying lesser interactions between the two.
Fig. 12.7 SEM micrograph

of the aggregates generated
by shear aggregation
Figure 12.2 shows the TEM and EDX characterization of graphene polyethylene
nanocomposite which was added with a small amount of chlorinated polyethylene
compatibilizer. As the graphene surface is polar as is the compatibilizer, there is a
possibility of positive interactions between them. On the surface of graphene, the
EDX analysis detected the presence of Cl atoms, whereas no Cl atoms were
detected in the matrix owing to its lower concentration. It confirmed the interac-
tions between the compatibilizer and the filler surface.
12.3 What is the Interaction of the Surface of Particles

with External Species?
The particles owing to their specific surface characteristics can interact with
external species. Figure 12.3 shows one such example of adsorption and desorp-
tion of tobacco mosaic virus molecules on the surface of functional polymer
particles [2]. The polymer particles are grafted with a thermally responsive
polymer, which leads to adsorption of the virus when the system is heated above
the lower critical solution temperature of the thermally responsive polymer
(Fig. 12.3a). On the other hand, when the system is cooled below the lower critical
solution temperature of the grafted polymer, the desorption of the virus molecules
takes place (Fig. 12.3b).
Fig. 12.8 Images

representing the adhesion of (a)
the surface grafted polymer
particles
500 nm
(b) 500 nm
12.4 How Does the Surface Interact with Charged Substrate,

or Stimulants Like Temperature, Salt, Sonication etc.?
Figure 12.4 depicts the microscopic characterization of the adsorption of nega-

tively charged polymer particles on the charged substrates. When the particles
12.4 How Does the Surface Interact with Charged Substrate 171
Fig. 12.9 TEM micrographs of the solgel-modified clay derivatives in acid-catalyzed

conditions using different TEOS/clay ratios: a 20/3, b 10/3, c 3/3 and d 1/3, and in non-
catalyzed conditions using different TEOS/clay ratios: e 10/3, f 10/5, g 5/5 and h 3/5. The arrows
indicate the mesoporous silica in bd and the silica nanoparticles in eh. Reproduced from [4]
were adsorbed on negatively charged disks, the adsorption was very poor owing to
the repulsion between the two (Fig. 12.4a). Heating of the disk to melt the polymer
particles also did not lead to the formation of a uniform thin polymer layer on the
surface. On the other hand, when the particles were adsorbed on the positively
charged disk, the adsorption was very efficient (Fig. 12.4b). A large number of
particles are seen to be adsorbed and the interstitial space between the particles
reduced significantly.
Similarly, the interaction of systems with stimulants like temperature, salt,
sonication etc. can be microscopically evaluated. Figure 12.5 shows the optical
micrographs of polymer particles grafted with thermally responsive
poly(N-isopropylacrylamide) polymer above and below lower critical solution
temperature (LCST) of 32 C [3]. Even after the collapse of grafted brushes at a
temperature above LCST, the particles do not aggregate and remain as indi-
vidual particles. Interaction of the particles with salt has been exemplified in
Fig. 12.6. SEM micrographs of polystyrene particles after swelling with
monomers followed by salt destabilization and polymerization have been shown.
The polystyrene particles were prepared both with and without surfactant. When
no surfactant was used (Fig. 12.6a), the polystyrene particles did not lose ori-
ginal morphology on swelling and destabilization, whereas, the particles gen-
erated with surfactant (Fig. 12.6b) completely lost their spherical morphology
and formed a continuous porous structure after swelling and destabilization.
Fig. 12.10 TEM images of a QD/MWNT = 1:10; b QD/MWNT = 1:1; c QD/MWNT = 10:1;
d HRTEM image of QD-MWNT (QD/MWNT = 1:1). Reproduced from [5] with permission
(Elsevier)
12.5 What is the Interaction Between Polymer Particles

Grafted by Another Polymer Layer?
Polymer particles on grafting with an adhesive polymer brush on the surface can
be made to aggregate/stick with each other. Figure 12.8 shows the TEM micro-
graphs of the bondingdebonding of the polymer particles grafted with adhesive
polymer brushes. The sticky nature of the brushes was obvious as on debonding
two particles, fibrous morphology of the brushes was resulted. The control on the
molecular weight of brushes on the surface can also lead to tuning of the aggre-
gation of the particles.
12.5 What is the Interaction Between Polymer Particles 173
Fig. 12.11 TEM micrographs of a magnetic nanoparticles (MNP), b multi walled carbon
nanotubes (MWCNT), c, d MNP/MWCNT hybrids. Scale bar in the images reads 100 nm.
Reproduced from [6] with permission (Elsevier)
12.6 How is One Inorganic Species Interacting

with the Surface of the Other (Decoration of the Surface)?
Figure 12.9 shows the TEM micrographs of the solgel-modified clays derivatives
[4]. In acid-catalyzed procedures, using high TEOS/clay ratio (Fig. 12.9a), clay
platelets are observed to be dispersed randomly in the mesoporous silica matrix.
On decreasing the TEOS/clay ratio, the silica morphology changes from the
Fig. 12.12 AFM images of silica substrates after soaking for 1 day in a single walled nanotube
dispersion (SWNT) prepared with 2 mL of ZrO2 solution and b the same SWNT dispersion with
an additional concentrated HCl amount. Reproduced from [7] with permission (ACS)
12.6 How is One Inorganic Species Interacting 175
Fig. 12.13 a, b TEM images of functionalized SWNTs after ferritin immobilization, c, d TEM
images of SWNTs after streptavidin-Au conjugates immobilization, e TEM image showing the
absence of protein immobilization. Reproduced from [8] with permission (ACS)
continuous cross-linked networks to the isolated mesoporous silica. In the non-

catalyzed conditions, the clay platelets are attached by silica nanoparticles, as
shown in Fig. 12.9eh. Figure 12.10 shows the TEM micrographs for the covalent
binding of quantum dots (QD) on the surface of carbon nanotubes [5]. Different
QD/MWNT mass ratio from 10:1 to 1:10 were used. It is observed that CdSe QDs
attached to a significant portion of the nanotube sidewalls. Increasing the amount
of quantum dots in the system, the extent of free dots also increased. Covalent
binding of magnetic nanoparticles on the surface of nanotubes is described in
Figure 12.15 shows the interactions of the silica particles with the polymer
colloids formed by emulsion polymerization as a function of functionalization of
silica as well as use of different initiators [10]. Figure 12.15a and b represented
uniform adsorption of silica particles on the surface of polymer particles, whereas
Fig. 12.14 a, b SEM images of gold nanoparticles immobilized on polymer-coated nanotubes

and c TEM image of gold nanoparticles immobilized on polymer-coated nanotubes. Reproduced
from [9] with permission (ACS)
such a structure was absent in Fig. 12.15c and d where colloidally stable system
could not be achieved. Figure 12.16 shows the magnetic nanoparticles in a com-
posite structure with a silica core, with Fe3O4 and gold as the inner and outer shells
Fig. 12.15 TEM images of: a polystyrene/silica nanocomposite particles, b poly(styrene-co-n-

butyl acrylate)/silica nanocomposite particles using a cationic AIBA initiator, c the identical
styrene homopolymerization in the presence of the same silica sol with an anionic APS initiator
and c AIBA-initiated styrene homopolymerization in the presence of a nonfunctionalized silica
sol (Bindzil 2040). Reproduced from [10] with permission (Wiley)
[11] by utilizing interactions of positively charged amino-modified SiO2 particles

for the assembly of negatively charged Fe3O4 nanoparticles. These then
Fig. 12.16 Top the preparation of three-layer magnetic nanoparticles. Bottom TEM images a,
b SiO2 particles covered with silica-primed Fe3O4 nanoparticles, c, d SiO2 particles covered with
silica-primed Fe3O4 nanoparticles and loaded with Au nanoparticle seeds and e three-layer
magnetic nanoparticles synthesized in a single-step process from the particles in c and
d. Reproduced from [11] with permission (ACS)
Fig. 12.17 TEM images of PSPVP thin films with Au nanoparticles in PVP lamellae after a
single cycle of Au nanoparticle synthesis: a plane view; b cross-sectional view. Reproduced from
electrostatically attract gold-nanoparticle seeds which subsequently lead to the

formation of a continuous gold shell upon reduction.
Figure 12.17 also describes the fabrication of electrically anisotropic multi-
layers of alternating polymeric layers and metallic layers with nanometer thickness
[12].
The molecular weight and composition of the copolymers control the size and
morphology of nanometer-sized domains. To functionalize one of the blocks of
copolymers for electronically, optically, or magnetically active nanodomains,
nanoparticles of metals can be incorporated selectively in nanodomains of diblock
copolymers.
12.7 How Does the Solvent Interact with the Morphology?
The interaction of solvent on the morphology is depicted in Fig. 12.18. SEM

image of the photonic crystal template is shown in Fig. 12.18a. The template is
coated with a polymer resin layer followed by its curing. Subsequently, the tem-
plate particles are removed by interaction with solvent leaving behind the porous
inverse opal structure as shown in Fig. 12.18b and c.

the a template of photonic
crystal (PC) template and b,
c the phenolic resin (PR)
inverse opal. Reproduced
from [13] with permission
(Wiley)
References 181
References
(2004)
2. Mittal, V., Matsko, N.B., Butte, A., Morbidelli, M.: Eur. Polym. J. 43, 48684881 (2007)
3. Kizhakkedathu, J.N., Norris-Jones, R., Brooks, D.E.: Macromolecules 37, 734743 (2004)
4. Qian, Z., Hu, G., Zhang, S., Yang, M.: Phys. B 403, 32313238 (2008)
5. Pan, B., Cui, D., He, R., Gao, F., Zhang, Y.: Chem. Phys. Lett. 417, 419424 (2006)
6. Xu, P., Cui, D., Pan, B., Gao, F., He, R., Li, Q., Huang, T., Bao, C., Yang, H.: Appl. Surf. Sci.
254, 52365240 (2008)
7. Zhu, J., Yudasaka, M., Zhang, M., Iijima, S.: J. Phys. Chem. B 108, 1131711320 (2004)
8. Chen, R.J., Zhang, Y., Wang, D., Dai, H.: J. Am. Chem. Soc. 123, 38383839 (2001)
9. Carrillo, A., Swartz, J.A., Gamba, J.M., Kane, R.S., Chakrapani, N., Wei, B., Ajayan, P.M.:
Nano Lett. 3, 14371440 (2003)
10. Schmid, A., Tonnar, J., Armes, S.P.: Adv. Mater. 20, 33313336 (2008)
11. Stoeva, S.I., Huo, F., Lee, J.-S., Mirkin, C.A.: J. Am. Chem. Soc. 127, 15362 (2005)
12. Yun, S.-H., Yoo, S.M., Sohn, B.-H., Jung, J.C., Zin, W.-C., Kwak, S.-Y., et al.: Langmuir 21,
36253628 (2005)
13. Li, H., Wang, J., Yang, L., Song, Y.: Adv. Funct. Mater. 18, 32583264 (2008)
Chapter 13
Nano to Micro and Macro
Characterization
13.1 Introduction
The commercial applications of materials often involve the structuring of nano-

particles into micro or macro structures. For example, the polymer particles are
generally structured to form monoliths which can then be used as chromatography
columns. Similarly, inorganic nanoparticles are fused together to form macro-
porous networks which can be used as catalyst supports or high strength and low
density metallic foams. Organic particles also form continuous films on the
substrates on which they are applied or coated. Characterization of such structures
for their porosity, surface roughness, uniformity as well as stability is required as
these characteristics drive the applications of these networks. A number of
examples describing these features are presented in the following sections.
13.2 What is the Morphology of the Porous Polymer

Network?
An example of porous polymer network formed by reactive gelation process is

demonstrated in Fig. 13.1 [1]. The polymer monoliths are generated by swelling of
the performed particles with a second batch of monomers followed by NaCl
gelation and polymerization. As is evident in the SEM images, both the networks
are porous in nature, however, the extent of fusion of the particles with each other
was different. It was owing to different chemical composition of the polymer
particles used to form the networks. Figure 13.2 also shows an example of network
formation (Fig. 13.2b) from the primary particles (Fig. 13.2a) by the action of
shear [2]. Thus, in this case, the particles were held together only physically and
there was no chemical bonding between them. Though the forces holding the
184 13 Nano to Micro and Macro Characterization
Fig. 13.1 SEM images of polymer monoliths generated by swelling of the performed particles
with a second batch of monomers followed by NaCl gelation and polymerization. a and c are the
low magnification images, whereas b and d are the same systems in high resolution respectively.
Reproduced from [1] with permission (Wiley)
networks together were only physical, but the networks were still stable over the
period of time. By controlling the weight fraction of the particles in initial sus-
pensions, the porosity of the resulting networks could also be controlled. Fig-
ure 13.3 also shows the differences in the network morphology at the surface and
in bulk. On the surface, the network structure is more closed and dense, whereas in
bulk, it is very porous. The wall thickness in the network also has variation.
An example of modification of monolith surface properties is demonstrated in
Fig. 13.4 [3], where SEM images of the monolith before and after 1 min grafting
with 2-acrylamido-2-methyl-1-propanesulfonic acid (poly(AMPS)) in water are
13.2 What is the Morphology of the Porous Polymer Network? 185
Fig. 13.2 a Primary polymer

particles and b resulting
physical aggregates generated
by the action of shear
shown. The SEM images did not exhibit any significant differences in morphology
of the poly(AMPS) modified monolith as compared to the parent monolith indi-
cating that the poly(AMPS) layer is rather thin. Figure 13.5 also depicts the mor-
phology of the epoxy resin based polymer monoliths as a function of reaction
Fig. 13.3 SEM micrographs

of a polymer network a on
surface and b in volume
temperature and molecular weight of porogen [4]. The change in the porosity of the
resulting networks confirmed the effect of porogen molecular weight and reaction
temperature. Figure 13.6 shows the SEM micrographs of the biporous bead (BiPB)
as well as microporous bead (MiPB) [5]. Compared to the smooth surface of MiPB,
the surface of the BiPB was rough, which indicated that there is significant difference
13.2 What is the Morphology of the Porous Polymer Network? 187
Fig. 13.4 SEM images of the monolith a before and b after 1 min grafting with 2-acrylamido-
2-methyl-1-propanesulfonic acid in water. Reproduced from [3] with permission (ACS)
in pore structure between these two types of beads. Figure 13.7 shows the SEM
micrographs of the monolith before and after grafting using a solution of 2-vinyl-
4,4-dimethylazlactone and divinylbenzene [6]. It was observed that the globular
structure within the original monolith is blocked upon grafting polymer along the
surface of the pores. Sheets and strands of the secondary polymer are clearly visible
which make the network impermeable when swollen.
Figure 13.8 also shows the scanning electron micrographs of poly(GMA-co-
TRIM) and poly(GMA-co-EDMA) monoliths [7], where GMA signifies glycidyl
methacrylate, EDMA is ethylene dimethacrylate and TRIM is trimethylolprophane
trimethacrylate. EDMA and TRIM are used as crosslinkers. It is observed that
though the pore sizes of both the TRIM- and EDMA-based monoliths are all large
enough, however, in the EDMA-based monolith, both the clusters and the voids
between clusters are much larger than those in the TRIM-based monolith.
Fig. 13.5 SEM images of epoxy based monoliths as a function of molecular weight of porogen
and reaction temperature. Reproduced from [4] with permission (ACS)
13.3 What is the Morphology of Polymeric Films on Substrates? 189
Fig. 13.6 SEM images of a, b biporous bead (BiPB) and c, d microporous bead (MiPB) at
different magnifications. Reproduced from [5] with permission (Elsevier)
13.3 What is the Morphology of Polymeric Films

on Substrates?
Figure 13.9 shows the SEM micrographs of the rough and smooth flat surface after
polymer modification [8]. The rough surface is observed to have presence of
uniform as well as porous polymer layer. The characterization of the micro-
structure of the film at different locations is investigated in Fig. 13.10 [9]. When
unheated, the polymer microstructure did not change as the polymer chains were
frozen owing to being below glass transition temperature. At a temperature near to
glass transition temperature, the polymer chains in the beads begin to collapse at
their locations and adhere to the adjacent beads. At temperature much higher than
glass transition temperature, the PS chains in the beads melt and adhere to the
adjacent beads rapidly. Figure 13.11 shows the effect of temperature on the for-
mation of thin polymer film on flat substrates [10].
The disc prepared at room temperature did not form a uniform polymer film as
the void spaces between the individual particles can be seen. The disc with 100 C
treatment indicates a better coverage as the particles have been fused with each
other thus filling the gaps initially present around them. However, a clear flowing
Fig. 13.7 Scanning electron

micrographs of the monolith
a before and b after grafting
using a solution of 2-vinyl-
4,4-dimethylazlactone and
divinylbenzene. Reproduced
from [6] with permission
(ACS)
of melt is visible in the disc subjected to a treatment of 150 C. The particles have
totally lost their structure and are totally melted and mixed with each other.
Performance of the polymeric coatings on substrates can also be characterized
through microscopy. Figure 13.12 shows the comparison of commercial paint and
polymeric-N-halamine containing paint, after 3 days of incubation in tryptic soy
broth [11]. As shown in Figure, a large amount of bacteria adhered onto the surface
of the film from commercial paint, forming microcolonies and developing into
biofilms. On the other hand, the polymeric-N-halamine containing paint film
showed no adhesion of bacteria and a much clearer surface was observed, indi-
cating biofilm-controlling capacity of the halamine containing paint.
Figure 13.13 also shows the SEM images of superhydrophobic polystyrene
films with special microsphere/nanofiber composite structures prepared via the
13.3 What is the Morphology of Polymeric Films on Substrates? 191
Fig. 13.8 Scanning electron

micrographs of a poly(GMA-
co-TRIM) and b poly(GMA-
co-EDMA) monoliths.
Reproduced from [7] with
electrohydrodynamics (EHD) method [12]. Both solution viscosity and the sur-
face-charge density strongly influence the morphology of the resulting nanofibers.
13.4 What is the Morphology of an Inorganic Macroporous

Network?
Figure 13.14 demonstrates SEM images of the n-C18H37NH3+, nanoscale silicate

platelets (NSP) dispersion and NSP/n-C18H37NH3+ (1:1 molar ratio) [13]. It is
evident from the SEM images that different degrees of surface roughness exist in
the samples. When single components are coated, the surface is relatively smooth.
On the other hand, a rougher surface is obtained by using 1:1 ionic equivalents of
NSP/n-C18H37NH3+.
Fig. 13.9 a SEM image for the rough (left) and smooth (right) flat substrate after polymer
modification and bd magnified images of the various locations of the polymer grafted rough
surface. Reproduced from [8] with permission (Wiley)
Figure 13.15 describes the formation of macroporous ceramics from particle

stabilized emulsions [14]. Microstructures of starting emulsions and final porous
ceramics prepared with various inorganic particles are shown in Fig. 13.15.
Particle-stabilized emulsions are formed by mechanical shearing of a mixture
containing the dispersed phase and concentrated suspensions of inorganic par-
ticles modified with short amphiphiles. The sintering of these emulsions led to
the formation of the macroporous networks of various inorganic materials.
13.5 What is the Photonic Crystal Morphology? 193
Fig. 13.10 AFM images of the film: a unheated film; b temperature near to Tg and c temperature
130 C. Reproduced from [9] with permission (ACS)
13.5 What is the Photonic Crystal Morphology?
Opals find their applications owing to uniformity in the structure. Thus, it is also
required to characterize if the uniform synthesis with long range order has been
achieved. Figure 13.16 shows an example of an inverse titania opal [15]. The
overall morphology of the opal is observed to be uniform.
Fig. 13.11 SEM images showing the effect of temperature on the film of adsorbed particles
when analyzed at a room temperature, b 100 C and c 150 C. Reproduced from [10]
Fig. 13.12 Comparison on the performance of commercial paint and polymeric-N-halamine

containing paint, after 3 days of incubation in tryptic soy broth. Reproduced from [11] with
permission (ACS)
13.5 What is the Photonic Crystal Morphology? 195

superhydrophobic
polystyrene films with special
microsphere/nanofiber
composite structures.
Reprinted from [12] with
permission (Wiley)
Fig. 13.14 SEM images of materials coated on glass and dried at ambient temperature: a
n-C18H37NH3+, b nanoscale silicate platelets (NSP) dispersion, c, d NSP/n-C18H37NH3+ (1:1
molar ratio) at different magnifications. Reprinted from [13] with permission (Wiley)
Fig. 13.15 Microstructures of starting emulsions and final porous ceramics prepared with
various inorganic particles. a, d alumina, b, e silica and c, f iron oxide Reprinted from [14] with
permission (Wiley)
References 197
Fig. 13.16 Scanning

electron micrograph of a
cleaved edge of an inverse
titania opal. Reprinted from
References
1. Mittal, V., Matsko, N.B., Butte, A., Morbidelli, M.: Macromol. React. Eng. 2, 215221
(2008)
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(2008)
3. Rohr, T., Hilder, E.F., Donovan, J.J., Svec, F., Frechet, J.M.J.: Macromolecules 36,
16771684 (2003)
4. Tsujioka, N., Hira, N., Aoki, S., Tanaka, N., Hosoya, K.: Macromolecules 38, 99019903
(2005)
5. Sun, G.-Y., Shi, Q.-H., Sun, Y.: J. Chromatogr. A 1061, 159165 (2004)
6. Tripp, J.A., Svec, F., Frechet, J.M.J.: J. Comb. Chem. 3, 216223 (2001)
7. Pan, Z., Zou, H., Mo, W., Huang, X., Wu, R.: Anal. Chim. Acta 466, 141150 (2002)
8. Sun, T., Wang, G., Feng, L., Liu, B., Ma, Y., Jiang, L., Zhu, D.: Angewandte Chemie. Int. Ed.
43, 357360 (2004)
9. Zhang, J., Xue, L., Han, Y.: Langmuir 21, 58 (2005)
10. Mittal, V., Matsko, N.B.: Open Surf. Sci. J. 1, 1419 (2009)
11. Cao, Z., Sun, Y.: ACS Appl. Mater. Interfaces 1, 494504 (2009)
12. Jiang, L., Zhao, Y., Zhai, J.: Angewandte Chemie. Int. Ed. 43, 43384341 (2004)
13. Lin, J.J., Chu, C.C., Chiang, M.-L., Tsai, W.-C.: Adv. Mater. 18, 32483252 (2006)
14. Akartuna, I., Studart, A.R., Tervoort, E., Gauckler, L.J.: Adv. Mater. 20, 47144718 (2008)
15. Mihi, A., Calvo, M.E., Anta, J.A., Mguez, H.: J. Phys. Chem. C 112, 1317 (2008)

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ISSN 1612-1317 ISSN 1868-1212 (electronic)

Library of Congress Control Number: 2012939481

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Springer is part of Springer Science+Business Media (www.springer.com)

1 Introduction to Microscopy Techniques. . . . . . . ............. 3

Part II Biological Related (Hydrated) Matter

2 Visualization of OrganicInorganic Nanostructures in Liquid . .. 13

2.8 Recent Advances: Cryo Analytical TEM (cryo ATEM). . . . . . 27

3 Macromolecular Distributions in Biological

4 Structure of the Biological Membrane (Detection

5 Structural and Analytical Chemical Analysis of the

6 Cellular Dynamics (Protein Transport,

7 Tomography of the Hydrated Materials . . . . . . . . . . . . . . . . . . . 85

Part III Polymer-Based Matter

8 Morphology in OrganicInorganic Composites . . . . . . . . . . . . . . 97

9 Interface Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 115

9.6 How Does the Morphology in the Blends Evolve

10 Surface and Volume Characterization . . . . . . . . . . . . . . . . . . ... 127

11 Confirmation of Surface Reactions . . . . . . . . . . . . . . ......... 147

12 Interactions Between Components. . . . . . . . . . . . . . . . . . . . . . .. 163

13 Nano to Micro and Macro Characterization . . . . . . . . . . . . . ... 183

1.1 Which is More Valid: Information Obtained by Eyes

V. Mittal and N. B. Matsko, Analytical Imaging Techniques for Soft Matter 3

1.2 Transmission Electron Microscopy

Conventional transmission electron microscopes [1] are electron optical instru-

1.2.1 Analytical TEM

The technique of electron energy-loss spectroscopy (EELS; Fig. 1.2) is of great

1.3 Scanning Electron Microscopy (SEM)

1.4 Atomic Force Microscopy

probe as it is scanned across the surface, a topographical image of the surface is

The characterization of colloidal systems like pharmaceutical or hydrated

V. Mittal and N. B. Matsko, Analytical Imaging Techniques for Soft Matter 13

Fig. 2.1 Cryo TEM of different hydrated systems. a nanoemulsion, b liposomes-proteins

2.2 Analytical Techniques Employed for the Characterisation

The characterisation of nano-sized colloidal systems requires diverse techniques

2.3 Transmission Electron Microscopy: Experimental Setup,

2.4 Cryogenic Transmission Electron Microscopy (Cryo

Generally speaking, the electron microscopic technique of choice for an artefact-free

Although water is the most abundant component of biological material, it is

without dilution. In general, dilution of the investigated nanoemulsions is advis-

formulations corresponded very well to the native structures as observed by cryo

2.5 Laser Light Scattering Versus Electron Microscopy

analysis are highly recommendable. A good overview of the investigated systems

2.6 Freeze-Etching and Freeze-Fracturing of Nanoemulsions

Freeze-etching, freeze-fracturing and cryo electron microscopy of frozen fluids are

Fig. 2.7 Freeze-fracture TEM micrographs of different lecithin-based nanoemulsions. Images

as chemical fixation, cryoprotective pre-treatment, cryofixation, freeze-fracturing,

2.7 Scanning Electron Microscopy, Cryo SEM and Freeze-

2.8 Recent Advances: Cryo Analytical TEM (cryo ATEM)

The most astonishing usage of EFTEM can be assigned to the determination of

At present, the description of biological ultrastructure is more closely related to the

V. Mittal and N. B. Matsko, Analytical Imaging Techniques for Soft Matter 31

sub-nanometer scale [1318], an ultimate resolution of biomolecule or organelle

molecule distribution and architecture) of the investigated biosample. Neverthe-

3.2 AFM Tuning Parameters Used for the Imaging

3.3 Dependence of the AFM Phase and Topographical

Fig. 3.3 AFM height image

in the phase image corresponds to ribosome agglomerates in the TEM image

3.4 Correlative AFM/TEM Analysis of the Protein

3.5 Identification of the Cell Constituents in AFM Phase