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Nanoscale
REVIEW

Comparative advantages and limitations of the basic

metrology methods applied to the characterization of
Cite this: Nanoscale, 2013, 5, 8781
nanomaterials
Pavel Linkov,a Mikhail Artemyev,ab Anton E. Emovcd and Igor Nabiev*aef

Received 8th May 2013
Accepted 12th July 2013
DOI: 10.1039/c3nr02372a
www.rsc.org/nanoscale
Fabrication of modern nanomaterials and nanostructures with specic functional properties is both
scientically promising and commercially protable. The preparation and use of nanomaterials require
adequate methods for the control and characterization of their size, shape, chemical composition,
crystalline structure, energy levels, pathways and dynamics of physical and chemical processes during
their fabrication and further use. In this review, we discuss dierent instrumental methods for the
analysis and metrology of materials and evaluate their advantages and limitations at the nanolevel.

1 Introduction its further progress strongly calls for advanced nano-

Nanotechnology is already a large industry and is expected to
continue to grow rapidly. Precise control of the sizes of objects
is the key issue of nanotechnology and nanoscience. The objects
they deal with are smaller than 100 nm and the necessary
precision for size measurement is oen as small as 0.1 nm
(Fig. 1). This requires new metrological approaches to be
developed, because the measurement techniques used for
conventional materials can rarely be directly applied to nano-
structures. The study of nanostructures and nanomaterials
requires special protocols that take into account the physical
phenomena that only occur in nanosized systems. Arrangement
of atoms or particles in nanostructures brings forth unusual,
sometimes exotic forms, such as fullerenes, coreshell nano-
particles, nanostructured metals and dendrites. This poses a
challenge for stereology and requires the development of
equipment that would permit the production of reproducible
nanostructures. Standards have to be set to match the advances
in the production and numerous applications of nano-
structures. Nanoscience has emerged at the interface of biology,
chemistry and materials technology, underpinned by physics
and rapidly developing computational materials science,1 and
a
Laboratory of Nano-Bioengineering, National Research Nuclear University, Moscow
Engineering Physics Institute, 31 Kashirskoe sh., 115409 Moscow, Russian
Federation. E-mail: igor.nabiev@gmail.com; Web: http://www.lnbe.mephi.ru
b
Institute for Physico-Chemical Problems, Belarusian State University, 220050 Minsk,
Belarus
c
Laboratory of Bionanotechnology, Shumakov Federal Research Center of
Transplantology and Articial Organs, 123182, Moscow, Russian Federation
d
SNOTRA LLC, 105318, Moscow, Russian Federation
e
European Technological Platform Semiconductor Nanocrystals, Institute of Molecular
Medicine, Trinity College Dublin, James's Street, Dublin 8, Ireland
Laboratory of Research in Nanosciences EA4682, Universite de Reims Champagne-
f including analysis of their structures, are generally considered
Ardenne, 51100 Reims, France
metrological techniques.
The importance of metrology for nanotechnologies is
obvious, since nanoscale structures cannot be manufactured
unless appropriate characterization methodologies are used.
Therefore, the ever-increasing applications of nanomaterials in
semiconductor technologies, photonics, optoelectronics,
biotechnology and chemical sensing drive an increasing
demand for nanometrology. One should measure a whole
number of physical characteristics to understand the physical
and chemical properties of nanoparticles and nanostructures.2,3
These parameters should also be monitored when nano-
particles are stored, because their changes with time in
dierent environments may be considerable and are mainly still
unknown. In addition, nanoparticles are oen coated with
surfactants or contaminants, these layers being not always
suciently characterized or even adequately identied.4
Many reports focus on the size and morphology of nano-
structures, paying less attention to the interplay between the
structure and function, as well as their dynamic nature. In this
respect, more problems arise from the fact that the thermody-
namics of nanoscale systems vary in dierent environments.
This adds real-time monitoring of the state of nanoscale objects
under various conditions and during various processes to the
list of challenges for nanometrology.5
Hence, metrology plays an increasingly important role in
nanoscience and nanotechnologies, with its development
promising both scientic discoveries and opportunities for
commercialization.
There is a consensus that the new functionalities oered by
nanoscale materials will give rise to a growing impact of nano-
enabled products over various markets.7 The development,
synthesis and characterization of novel nanomaterials,
critical for advancement of nanotechnology. This requires real-

This journal is The Royal Society of Chemistry 2013 Nanoscale, 2013, 5, 87818798 | 8781
Nanoscale
Fig. 1 The ISO classication of nanoobjects. Included as nanoobjects are nanoparticles (nanoscale in all three dimensions), nanobers (nanoscale in two dimensions)
and nanoplates or nanolayers (nanoscale in only one dimension). * The term nanoscale refers to a size between 1 and 100 nm. Copyright 2011, Wiley.6
time monitoring of the synthesis and manipulation processes at
the nanoscale. Therefore, nanometrology and suitable charac-
terization techniques are of paramount importance for further
progress in nanotechnology.8
In this review, we discuss the principles, applications,
advantages and limitations of the following instrumental
methods for characterization of nanomaterials:

Electron microscopic techniques, including
- Transmission electron microscopy (TEM)
- Scanning electron microscopy (SEM)

Scanning probe microscopic (SPM) approaches, including
- Atomic force microscopy
- Scanning tunneling microscopy (STM)

X-ray methods, including
- X-ray photoelectron spectroscopy (XPS)
- X-ray diraction (XRD)

Optical spectroscopic techniques, including
- Absorption spectroscopy
- Dynamic light scattering (DLS) or photon correlation
spectroscopy (PCS)
- Fluorescence correlation spectroscopy (FCS).
ADF, annular dark-eld imaging; AFM, atomic force microscopy; BF, bright-eld; density, can be used to draw an electron density map. Other-
CSD, Cambridge structural database; DF, dark-eld; DLS, dynamic light scattering;
EDS, energy dispersive spectroscopy; EELS, electron energy-loss spectroscopy; EFM,
electrostatic force microscopy; ESCA, electron spectroscopy for chemical analysis;
ESEM, environmental scanning electron microscopy; EPMA, electron probe
microanalyzer; EXAFS, extended X-ray absorption ne structure; FCS, uorescence
correlation spectroscopy; FIB, focused ion beam microscopy; HRTEM,
high-resolution transmission electron microscopy; ICDD, international centre for
diraction data; LVEM, low-voltage electron microscopy; NIST, National Institute
of Standards and Technology; MFM, magnetic force microscopy; PCS, photon
correlation spectroscopy; SAXS, small angle X-ray scattering; SEM, scanning
electron microscopy; SESSA, simulation of electron spectra for surface analysis;
SPM, scanning probe microscopy; STEM, scanning transmission electron
microscopy; STM, scanning tunneling microscopy; TEM, transmission electron
microscopy; TOPO, tri-n-octylphosphine oxide; WAXS, wide angle X-ray scattering;
XPS, X-ray photoelectron spectroscopy; XRD, X-ray diraction.
8782 | Nanoscale, 2013, 5, 87818798
Review
A summary of comparative advantages and limitations of the
basic metrology methods is presented in Table 1 and the details
of applications of these methods to the characterization of
nanomaterials are discussed below.
2 Electron microscopic methods
Determination of the structure is essential for nanomaterial
research. TEM and SEM are the most popular electron micros-
copy techniques that can be used to characterize the structural
and surface properties of nanomaterials either directly or
indirectly.911 These techniques dier from one another in
surface sensitivity. The choice of the technique is determined by
what information about the material is to be obtained (Fig. 2).
Electron microscopy yields two-dimensional images. The
contrast mechanism is based on the scattering of electrons.
Traditionally, sizes are measured in electron microscopic
images by applying the intensity threshold uniformly over the
image. Simple analysis allows the area and radius of the particle
to be determined and the size histogram to be plotted.
If the sample is suciently thin, the modulation of electrons
transmitted through the sample, depending on the electron
wise, the sample is raster-scanned with a nely focused beam,
and the reected electrons are used to form its topographical
image.
2.1 Transmission electron microscopy
TEM is a tool for structural and chemical characterization with
a high spatial resolution.9,12 Modern TEM can directly produce
images of atoms in crystalline specimens at resolutions close to
0.1 nm (smaller than the distance between atoms), which
permits quantitative chemical analysis of a single nanocrystal.
This type of analysis is especially suitable for the range of sizes
from atomic ones to hundreds of nanometers. TEM can be used
This journal is The Royal Society of Chemistry 2013
 Table 1 An overview of basic methods for nanometrology

Methods Nanosystem analyzed Parameter measured Resolution Problems Ref.

Review

Electron Transmission electron Clusters, nanocrystals, Morphology (particle size and shape); Lateral resolution, 0.1 The thickness limit is 200 nm. The 953
microscopy microscopy (TEM) nanoparticles, quantum crystallographic information (detection nm method entails time-consuming
methods dots, dendrimers, of atomic scale defects); compositional sample preparation to make it
nanowires, nanotubes, and information (the elements and thin; the eld of view is relatively
biomolecules compounds the sample is composed of); small; the sample may be
information on the phases present damaged by the electron beam,
(lattice spacing measurement); particularly in the case of
topography and elemental mapping biological materials; ultrahigh
(STEM) vacuum is required for atomic-
scale resolution, light atoms are
insuciently contrast
Scanning electron Atomic at surfaces, Topography (topography map, surface Depth, 0.55 nm; lateral SEM works only with conductive 5480
microscopy (SEM) nanoparticles and characteristics); morphology (particle resolution, 120 nm samples. The method requires
nanocrystals, multilayered shape and size); composition (elements high vacuum; the examination of

This journal is The Royal Society of Chemistry 2013

nanowires, carbon and compounds); crystallographic wet materials and biological
nanotubes, nanobers, and information (the arrangement of atoms) samples is dicult
biomolecules
Scanning probe Atomic force Both conductive and non- Topography, morphology, elasticity of Depth, 0.55 nm; lateral The single scan image size is 8196
microscopy microscopy (AFM) or conductive nanomaterials. the surface, frictional characteristics, resolution, 0.210.0 nm small. The AFM scanning speed is
methods scanning force Nanocrystals, nanotubes, specic molecular interactions and a limitation. The relatively slow
microscopy (SFM) and biological samples magnetic characteristics of the surface, rate of scanning during AFM
total density of (valence-) electron states imaging oen leads to a thermal
up to the Fermi level at the surface dri. AFM images may be aected
by hysteresis of the piezoelectric
material
Scanning tunneling Only conductive including Surface topology (size, shape, roughness, Depth, 0.55 nm; lateral STM works only with conductive 97115
microscopy (STM) semiconductor particles: defects, electronic structures and local resolution, 210 nm samples. It requires minimization
nanocrystals, thin lms, density of states). Surface properties, of all electrical and mechanical
nanotubes and molecules surface structures and surface reactions noises
and single point defects
X-ray methods X-ray photoelectron Nanoparticles, nanowires, Qualitative and quantitative elemental Depth, 0.510 nm; Considerable errors in analyzing 126156
spectroscopy (XPS) thin lms, composition in the top 110 nm surface lateral resolution, 5 chemically heterogeneous
semiconductors, polymers, layers. The chemical state identication nm50 mm surfaces; degradation during
and biomaterials of one or more of the elements in the analysis; the method requires
sample. The binding energy of one or high vacuum
more electronic states. The density of
electronic states
X-ray diraction (XRD) Nanoparticles, virus Surface topology, crystallographic Peak broadening at small crystal 157189
particles, polymers, thin information (the type of the crystal sizes makes it dicult to
lms, and biomolecules structure, interplanar dierences, determine the crystalline
ensemble spatial orientation, and charge structure of nanoparticles
distribution around atoms). Crystal
structure, chemical composition and
physical properties of materials and thin
lms

Nanoscale, 2013, 5, 87818798 | 8783

Nanoscale
 Nanoscale Review

190210

211234
Ref.

they absorb it; hence, the data are

Solid particles in a heterogeneous

intensity-biased towards larger or

towards more refractile particles

sample. In samples with an NP
sample scatter light more than

doubtful. Restriction to turbid

Average particle size, which is

mixture, analysis gravitates

contaminant particles in a
Problems

samples
0.5 nm micrometer-
Resolution

range

hydrodynamic radius of macromolecules

Volume concentration of the substance.

macromolecules or micelle equilibrium

particle diusion. The Stokes radius or
Indirect determination of particle size

hydrodynamic radius of particles; the

Particle size distributions, the rate of

or block copolymer micellar systems;

changes in the conformation of

Fig. 2 Schematic of the interactions of dierent types of electron and ion beams
with a sample and the types of signals that can be generated. Copyright 2012,
Parameter measured

Elsevier.12

to obtain information on the particle size, shape, crystallinity

and interparticle interaction. TEM also allows diraction
patterns to be identied that also contain crystallographic
gels, amino acids, proteins,

information.13,15
solutions, polymer melts,
suspensions, thin lms,

The resolution limit of the modern TEM with the use of

Colloidal suspensions,
Nanosystem analyzed

aberration-corrected instruments is about 0.05 nm. The reso-

Colloidal solutions,

and liquid crystals

micelles, polymer

lution of TEM is generally dened as the performance obtain-

able with ideal specimens, i.e., those suciently thin to avoid
and powders

chromatic eects.14 These are related to the chromatic aberra-

tion of the electrons that have lost energy when passing through
the specimen. A thick specimen may reduce the attainable
resolution to 1.52 nm. Regarding nanoparticles, these eects
may occur if a particle preparation is too dense; a preparation of
scattering (DLS) and

well-dispersed particles on a thin support does not normally

spectroscopy (FCS)

cause a serious loss of resolution.

Dynamic light
spectroscopy

TEM is used to analyze the morphology of crystal and crys-

uorescence
Absorption

correlation

tallite electrode materials, including metals,16 oxides,17 semi-

Methods

conductors18,19 and electrochemically produced metal

nanopowders.20,21 Dark eld (DF) and bright-eld (BF) images
can be used to control the granulometry and dispersion of
(Contd. )

nanomaterials. For example, amorphous and crystalline

Optic methods

domains of composite SnC anodes of lithium batteries are

easily detectable. TEM has been reported to reveal dierences in
Table 1

the size of tin particles at the surface and in the bulk material.22
Combination of dierent modes of TEM with other techniques

8784 | Nanoscale, 2013, 5, 87818798 This journal is The Royal Society of Chemistry 2013
Review
of morphological and structural analysis, such as SEM or XRD,
permits a complete characterization of composite materials.23,24
TEM is widely used for investigation of biological objects,
including viruses, bacteria and individual biomolecules. Modi-
cation of biomolecules with metal nanoparticles made them
easily detectable by means of TEM. The combined use of TEM
and labeled antibodies provides a powerful tool for locating
bacterial and viral antigens both on the surface of bacteria or
viral particles and in thin sections of infected tissues.
TEM identication of antigens is potentially useful in diag-
nosis of viral gastroenteritis, in examining immunocompro-
mised patients25 and in the cases of new infectious agents,
exotic viral infections. TEM is also suitable for investigation of
the core antigen of the hepatitis B virus,26 analysis of viral
capsids and study of the cytopathic eect in cell cultures.25
Below we describe some specic types, modes and applications
of TEM.
Transmission electron microscopes equipped with a eld
emission source and a high-intensity probe beam permit
elemental analysis in specimen areas no thicker than several
hundred angstroms with a high spatial resolution (1 nm).
Since TEM oers a resolution down to atomic sizes, it can
provide useful information about the chemical composition of
the specimen. Scanning transmission electron microscopy
(STEM), a variant of TEM, is used for this purpose.
Let us briey consider the dierent variants of TEM used in
nanoscience.
Scanning transmission electron microscopy. In scanning
transmission electron microscopes, as in conventional trans-
mission electron microscopes, electrons pass through a su-
ciently thin specimen. The dierence of STEM is that an
electron beam is focused into a narrow spot and rastered over
the specimen. This makes it suitable for analytical techniques,
such as energy dispersive X-ray spectroscopy (EDX or EDS)
mapping, electron energy-loss spectroscopy (EELS), Z-contrast
and annular dark-eld imaging (ADF).2729 These signals can be
obtained simultaneously, thereby allowing direct correlation of
image and quantitative data. The approach is especially useful
for deciphering the structure of polycrystalline and multiple-
phase specimens. STEM-based elemental analysis utilizes
inelastic scattering (loss of energy) of the electron beam passing
through the sample. EDS30 and EELS31,32 techniques based on
the detection of either characteristic X-rays emitted by the
specimen or inelastically scattered electrons, respectively, are
the most common techniques used for this purpose.33
In EDS the characteristic X-rays emitted by various elements
when incident X-rays or electrons hit the specimen are analyzed.
Using state-of-the-art ultra-thin or windowless detector
elements, atoms down to boron can be detected.34 EDS is used
in failure analysis to detect various elements in defects.35
Electron energy loss spectroscopy. During the inelastic
scattering, transmitted electrons lose energy, the amount of
which is element-specic.33,36 As a result, the transmitted beam
consists of electrons with a range of energies. In EELS, a spec-
trometer placed underneath the electron microscope column is
used to detect electrons with the energy loss that is character-
istic of the element detected. EELS oers a higher spatial
This journal is The Royal Society of Chemistry 2013
Nanoscale
resolution (1 nm) than EDS and the possibility to detect light
elements (e.g., C, O and N).
Z-contrast scanning transmission electron microscopy
(Z-contrast STEM). Z-contrast scanning transmission electron
microscopy (Z-contrast STEM) has been proven to be a valuable
tool for identifying chemical elements in materials, where the
contrast is directly related to the atomic number (Z-contrast
image).29,37
Erni et al.38 reported on dierentiation between elements in
AlAg precipitates by means of Z-contrast STEM.
ADF is particularly suitable for analysis of biological
samples.39 Here, STEM is more ecient than conventional TEM
because it ensures high-contrast imaging of macromolecular
complexes without staining.4042
High-resolution transmission electron microscopy
(HRTEM). High-resolution transmission electron microscopy
(HRTEM) is a powerful tool for crystal structure imaging and
structure analysis. Modern high-resolution microscopes make it
possible to resolve inter-atomic distances and to image indi-
vidual atoms and crystalline defects. Views of a crystal taken
from dierent angles can be combined to obtain a 3D map. This
technique is called electron crystallography.
The highest resolution attained thus far is 0.047 nm. It was
obtained at the Tokyo Institute of Technology with the use of a
JEOL R005 double aberration-corrected electron microscope
equipped with a cooled eld-emission gun.43,44 O'Keefe and
Shao-Horn45 obtained a similar resolution (0.05 nm) using a
new design of the aberration corrector, which reduced spherical
aberrations. The use of HRTEM allows a researcher to deter-
mine the positions of atoms in materials, which has made it an
indispensable tool for research and development in many elds
of nanotechnology, including heterogeneous catalysis and the
semiconductor devices for electronics and photonics.37,46
A diculty with HRTEM is that the image formation relies
on phase contrast, which is not always intuitively interpretable
because of the inuence of strong aberrations in the imaging
lenses of the microscope.
HRTEM combined with other methods has been used to
study the structural properties of carbon nanotubes, including
their diameter, chirality and number of layers, as well as to
detect defect sites in them.47,48
A new method has been reported that combines AFM
measurements with independent analysis using HRTEM. This
approach yielded important information on the nanotube
structure, the number of layers, the interlayer distance and the
location of encapsulated molecules.49 The use of TEM grids with
thin carbon membranes allows specimens to be transferred
between the AFM and HRTEM systems. Note that this approach
is not limited to specic types of nanostructures.
Thus, HRTEM is a unique technique for direct imaging of
individual particles, including high-resolution analysis of their
surfaces in a side view.50,51
HRTEM has been used as a supplement to diraction
analysis for determining the structure of liquid crystalline
polymers, including a frustrated smectic-A, aperiodic crystals
formed from the nematic phase and macromolecular assem-
blies consisting of cubic structures. In the same study, direct
Nanoscale, 2013, 5, 87818798 | 8785
Nanoscale
examination of defects has been performed for various liquid
crystalline phases.52
A comprehensive review of HRTEM studies of nanocrystals
has been published recently by Zhou.53
Limitations of TEM and HRTEM. Many materials require
extensive sample preparation to produce a sample thin enough
to be electron transparent, which makes TEM analysis a rela-
tively time consuming process with a low throughput of
samples. Also, the eld of view is relatively small, raising the
possibility that the region analyzed may not be characteristic of
the whole sample. There is probability that the sample may be
damaged by the electron beam, particularly in the case of bio-
logical materials (introduction of possible artifacts). Generally,
the heavier atoms scatter electrons much more eectively than
the lighter atoms. Hence, the metal particles are signicantly
more clearly dened than the polymer particles. In addition, if
the particulates are composed of crystalline domains, their
orientation will also aect the image contrast.
2.2 Scanning electron microscopy
SEM is among the most versatile and widely used techniques for
surface analysis, allowing the study of both morphology and
composition of various materials. This technique is popular
because it is relatively quick, inexpensive and essentially
nondestructive. It has many applications in biology, as well as
in semiconductor research, study of catalysis and other mate-
rials sciences. There are many types of scanning electron
microscopes specially designed for various elds of research,
including routine morphological studies, high-speed composi-
tional analyses and the study of environmentally-sensitive
materials. The main advantages of SEM are a high lateral
resolution (110 nm), large focus depth and the possibility to
use various electronspecimen interactions for both imaging
and chemical analyses. In contrast to TEM, SEM does not
impose strong restrictions on the specimen size and does not
require elaborate preparation of specimens.9,54
Typical scanning electron microscopes with a eld emission
system and an in-lens detector provide as large as 400 000
magnication and a resolution of about 1 nm.
SEM is suitable for the morphological and compositional
analyses of inorganic nanocrystals and electrochemical power
sources.55,56 The electrode morphology can be studied at
dierent magnications to estimate the homogeneity,
compactness and granulometry of the sample. In addition,
analysis of backscattered electrons provides information on the
sample composition and homogeneity. By combining SEM with
an electron probe microanalyzer (EPMA), one can perform
elemental qualitative and quantitative analyses of very narrow
areas of the sample.57
Owing to the progress in computer technologies, SEM has
become easier today. This line of development of electron
microscopy originated in the semiconductor industry.58,59 SEM
is used there for automated measurement of critical dimen-
sions of semiconductor wafers. The optimum detector strategy
and microscope settings considerably vary for dierent tasks
and should be specially selected for each particular case.
8786 | Nanoscale, 2013, 5, 87818798
Review
The main limitations of conventional SEM are related to the
requirement of high vacuum and electroconductivity of surfaces
to yield high-magnication SEM images. It means that biolog-
ical samples and other wet samples cannot be studied directly.
Water should be either removed or xed during the preparation
of specimens, which increases the probability of artifacts and
samples must be coated with an ultrathin coating of conducting
materials, generally using heavy metals. It may obscure some
details of the ne structure of the surface, although the use of
microscopes equipped with a eld emission gun partly
compensates for this drawback. Sputtering or various vacuum
deposition techniques are used for sample coating with
carbon,60 gold61,62 or alloys.60,63 However, such pretreatment is
complicated and may damage or deform the sample. An alter-
native is using ionic liquids, which remain molten even at room
temperature and do not vaporize even under vacuum. Ionic
liquids may apply to SEM visualization of hydrous samples;64,65
in this case the samples do not contract much even aer drying.
The variant of SEM called environmental scanning electron
microscopy (ESEM) makes it possible to examine directly objects in
the wet mode and in the presence of air (at a low pressure) without
high vacuum and complicated preparation of specimens. With
this new technique, many materials can be imaged in an unaltered
state as an alternative to metal coating. In addition, one may alter
the environment of the sample so as to trace, e.g., hydration and
dehydration of the material in the sample chamber.66,67
This also applies to biological samples, which can be
examined in their native state, which makes ESEM especially
attractive for biomedical research68 and bioengineering, in
particular, for analyzing the interactions between biomaterials
designed for tissue engineering.69
Detailed comparison of ESEM and high-vacuum SEM in
terms of studying the cell morphology is presented in ref. 70.
Finally, ESEM is sensitive to the electronic structure of the
sample, because it does not require a surface coating and
employs a specic mode of cascade amplication.71
There are two major drawbacks of ESEM. The rst one is
setting the threshold level, which may not be easy for poorly
contrasting particles (e.g., small particles of polymers) and inho-
mogeneous materials. The other drawback is that it is dicult to
estimate accurately the sizes of agglomerated particles, because
the lack of a considerable dierence in intensity makes a simple
threshold insucient to discern individual particles.
Focused ion beam (FIB) microscopy. This technique, strictly
speaking, should not be called electron microscopy, although
its principle is the same as that of SEM. The dierence is that
beams of ions (usually gallium) are used instead of electron
beams. Since ions have much larger masses than electrons, they
cause sputtering of secondary and backscattered electrons, ions
and atoms.72 The abundance of their signals ensures a very
high-contrast imaging.7375
The use of helium instead of gallium oers several specic
advantages that ensure a higher resolution of imaging
compared to conventional SEM.76
In addition to better resolution, FIB also provides unique
mechanisms of contrasting that facilitate discrimination and
identication of materials.74,77
This journal is The Royal Society of Chemistry 2013
Review
Low-voltage electron microscopy (LVEM). LVEM combines
SEM, TEM and STEM in a microscope operating at a low electron
accelerating voltage (5 kV).78 The low voltage enhances the
contrast of imaging, which is especially important for analysis of
biological samples.79 With the enhanced contrast, there is less, or
no, need for staining. The slices generally should be thinner than
in the case of conventional STEM (2070 nm). LVEM has been
used, e.g., to study thin lms of pentacene (an organic semi-
conductor).80 In that study, highly contrast images of very thin
lms of a thermally evaporated material were obtained. The
resolution of the method allowed the authors to discern a poly-
crystalline layer of the thin-lm phase, over which another layer of
crystals was nucleating and growing.
3 Scanning probe microscopy
3.1 Atomic force microscopy
AFM is the most popular representative method of a family of
techniques collectively called SPM that are used to analyze
structures and processes on the atomic scale.
AFM measures the sample surface topography with a reso-
lution of several nanometers and visualizes the orientation and
spatial arrangement of nanostructures on surfaces.
AFM provides an atomic resolution under certain condi-
tions; surface topography is usually resolved suciently on the
nanometer and even angstrom scale in the vertical direction,
while the lateral resolution of AFM is lower. The AFM lateral
resolution depends on the tip shape, the scan domain and the
number of measurements per scan. The vertical resolution is
determined by the piezoelectric crystal moving the specimen
relative to the tip and the deection of the detecting cantilever.
An advantage of AFM is that it does not require staining, con-
trasting with special agents, or conductive coatings. AFM
specimens are relatively easy and quick to prepare. The tech-
nique is nondestructive, so that the same sample can be used
for multiple analyses.81,82
AFM is suitable for biological studies at the molecular level,
such as analyses of cell membrane structure and protein
conformations, as well as DNADNA interactions and enzymatic
reactions.8487 SPM is also used to measure the mechanical
properties of single molecules, molecular ensembles (including
intracellular structures) and biomolecule interactions.88,89 The
best AFM sensitivity in measuring the attraction and repulsion
forces is about several piconewtons. The technique has been
used to measure the adhesion force at the level of single
cells.88,90 In one of these studies,91 a so-called nano-picker was
fabricated out of an AFM cantilever by the nanoetching method;
the cell interaction force is measured by the deection of the
nano-picker. The adhesion that may occur between the tip and
the substrate can also be measured.
AFM can be used to manipulate nanoobjects, e.g., to move
carbon nanotubes and bend them into a desired shape, by
mechanically pushing them with the probe tip.92
The detection of polyacrylic acid regions in a poly(ethylene
glycol) matrix by means of AFM imaging has been reported.93
The methods for the control of surface chemistry at the
nanoscale by means of AFM are reviewed in ref. 94.
This journal is The Royal Society of Chemistry 2013
Nanoscale
Like other microscopic techniques, AFM has inherent limi-
tations. Imaging artifacts are especially characteristic of the
tapping mode of AFM, where the cantilevered tip touches the
surface at a frequency of several kilohertz (thus serving as an
oscillating probe). The nite tip size and the thermal dri of the
scanner95 are sources of inevitable bias of the mapped surface
topography. Hence, one should be careful when interpreting the
maps.
An example of a bias in the estimation of nanoparticle sizes
using AFM tapping mode is a study on measuring the diameter
of single tungsten oxide particles.96 The estimated nanoparticle
size was found to depend on the oscillation amplitude of the tip,
as well as the physical properties of the surface of the material
(individual particles and the sample as a whole), elasticity and
surface energy and on the curvature of the tip.
3.2 Scanning tunneling microscopy
STM, another variant of SPM, is also an essential tool for
atomic-scale examination of the electronic and topographic
structure of a wide range of materials. It is used in biology,
microelectronics and materials science.97
Like AFM, this technique makes it possible not only to
visualize the surface topography and measure the surface
properties, but also to manipulate atomic-scale structures and
initiate reactions on the surface of the sample. The STM tech-
nique allows one to move pieces of material as small as single
atoms and place them at specic points on the surface, thereby
designing novel nanosized structures.98100
The fundamental dierence of STM from conventional SEM
is that it employs tunneling energies, which are substantially
lower than electron energies. Therefore, STM is suciently less
prone to topological artifacts, which, in SEM, result from the
interaction of high-energy electrons with the sample substrate.
STM can produce atomic-scale images in various media, from
vacuum to liquid.101 The technique uses direct image contrast
not requiring dyes or contrast agents. However, it can only deal
with conductive samples.102
In many cases, STM is indispensable in direct monitoring of
atomic and molecular dynamics and chemical reactions,
including catalysis and self-assembly processes. STM oers an
unprecedented new insight into the mechanisms of catalysis;
studies using this technique have revealed the specic cata-
lytical roles of individual surface sites, e.g., edges and surface
defects (kinks and atom vacancies).103
Fast-scanning STM allows the visualization of dynamic
processes on surfaces in the form of a series of time-resolved
images (STM movies).104,105
STM employs dierent mechanisms to move nanoobjects.
The main three ways to manipulate single molecules or atoms
are based on (a) the control of the tipadsorbate interaction
force, (b) the application of an electric eld and (c) the use of
tunneling electrons.106108 It has been demonstrated that the
most eective results were obtained if dierent STM manipu-
lation techniques are combined together. This approach has
been used, e.g., to induce a sequence of chemical reactions.109
STM-based manipulations have also been used in a molecular
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Nanoscale
design method where chemical bonds in large molecules are
selectively broken.110 It is also possible to construct organic
molecules out of hydrocarbon fragments as if they were
building blocks.111,112 The STM manipulations used to move
molecules or atoms across the surface are referred to lateral
manipulations.106 There is also a procedure called vertical
manipulation, in which the molecular building blocks are
moved from the probe tip to the substrate or vice versa. Here, the
transfer is driven by an electric eld, voltage pulses, or
mechanical force (when the tip is in direct contact with the
object to be moved).106,112
When STM is used for examining semiconductor samples, the
tunneling current contains information on the local densities of
states of the specimen. Therefore, STM not only provides detailed
images of the surface and data on the nanostructure morphology,
but also allows the electronic properties of the material to be
measured. As shown in Fig. 3, the high resolution of the micro-
scope is sucient for obtaining STM images of single point
defects even below the surfaces and characterization of their
electronic structure, charge carrier exchange between the dierent
defect levels and conduction/valence bands.113
STM data on the adsorption of small biomolecules,
including amino acids114 and nitrous bases,115 have provided
insight into the mechanisms of their interaction and assembly
into proteins and nucleic acids, for example, a clear image of a
DNA double helix as a well-ordered co-adsorption structure.
Finally, STM was approved to be an indispensable technique
for investigation and quantitative characterization of surface
phenomena and structural and functional parameters on the
atomic scale, including local bonding geometries, defects,
nonperiodic structures and coexisting complex phases.
3.3 Scanning probe nanotomography
In recent years, characterization of local nanoscale 3D volume
organisation of complex nanomaterial systems becomes a more
and more demanding task. For tomography analysis of three-
dimensional nanostructures in the bulk of materials, SPM as a
surface characterization technique, has to be combined with a
corresponding technique for removal of the surface material to
access the structures beyond the surface. Then 3D volume data
can be generated by stacking of consecutive 2D SPM images
Fig. 3 An STM image and virtualized image of a H-passivated Si (100) 2  1 reconstructed surface, showing H-passivated dimers, vacancies and dangling bonds (or
contamination) on both terraces. Copyright 2012, Elsevier.83
8788 | Nanoscale, 2013, 5, 87818798
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acquired from the studied surface area aer the removal of
controlled sequential thin layers. Resolution of 3D nano-
tomography imaging in the depth direction therefore will be
determined by the minimal repeatable thickness of the layer
removed. Until now two main approaches of the sample surface
processing have been eectively used for SPM nanotomography.
The rst approach is based on in situ automated wet chem-
ical etching116 or plasma etching117 of the sample surface fol-
lowed by imaging of the etched surface. This 3D imaging
approach has been applied for volume reconstruction of the
phase distribution in dierent polymer blends and human
bone.116118 A repeated removal of 7.5 nm thick surface layers
was reported. Even harsh sample processing like low pressure
plasma, solvent vapor treatments under high electric elds and
other aggressive treatments inside the SPM without moving the
sample are possible with the developed quasi in situ scanning
probe microscope system.119
A main disadvantage of this etching-based approach is the
potentially dierent etching rates of the various components or
phases in complex heterogeneous or hybrid so matter systems
such as blends, nanocomposites with hard materials or crys-
talline-amorphous phases. Such dierences in etching rates
distort the actual 3D structure in the reconstructed volume and
cause uncertainty in Z-resolution of the technique.
Another methodology and instrumentation approach for
scanning probe nanotomography combines SPM with an
ultramicrotome.120 This hybrid apparatus enables direct SPM
measurements of a sample block face surface immediately aer
each sectioning by an ultramicrotome knife. The thickness of
consecutive sections can go down to 12 nm. The block face
surface can be analyzed by a plethora of SPM modes including
phase imaging, current distribution measurements, electro-
static force microscopy (EFM) and magnetic force microscopy
(MFM), to name a few. Therefore, in this way, one can study not
only the 3D morphology organization of the sample but also the
volume distribution of local mechanical, electrical and
magnetic properties. This technology was successfully applied
for studying the 3D structure of biological objects and materials
and dierent advanced materials like polymer blends, nano-
composites and nanohybrid systems. One of the advantages
of this approach is that it has no general limitation on the
number of sections performed and overall thickness of the
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reconstructed volume; analysis of several micrometer thick
volumes is practicable.
The study of polymer/nanotube121 and polymer/graphene
conductive nanocomposites122 by SPM tomography with the use
of conductive AFM measuring mode enabled us to reveal the
local nanoscale organisation and connectivity of 3D conductive
networks of carbon nanotubes and graphene sheets corre-
spondingly, which determines the macroscopic properties of
such nanocomposites. Such conductive 3D networks could not
be investigated by any other microscopy modality developed
until now.
SPM nanotomography analysis of liquid crystal/quantum dot
nanohybrid systems has proved that the technique enables us to
control 3D distribution of nanoparticles in a matrix and inves-
tigate the inuence of nanoparticle distribution on local 3D
organization of the matrix structure.123,124
The range of so matter systems that can be analysed by SPM
tomography may be signicantly broadened with a new design
of a hybrid system for low temperature measurements, which
has been introduced recently.125 This system incorporates a cryo
AFM directly mounted in the cryogenic chamber of the ultra-
microtome, which permits in situ measurements of the sample
surface aer cryo sectioning without any modication of the
sample ultrastructure or transfer of the sample. Such an
approach provides unique indispensable capabilities for
investigation of native structures of so polymers, hydrated
materials and biological systems stabilized by cryoxation,
which cannot be properly sectioned and studied by AFM under
ambient conditions.
To date cryo SPM nanotomography has been used to study
unmodied nanoscale structures of so polymer systems like
synthetic acrylonitrile/butadiene copolymer latex rubbers and
polyamide 6/styreneacrylonitrile composites.
Further development of SPM nanotomography techniques is
dedicated to provide new insight into 3D organisation of
nanocomposites, biomedical materials (including pharmaceu-
tical preparations and drug delivery systems), and biological
objects (cells, their organelles and tissues).
4 X-ray methods
The wavelengths of X-rays (from 0.01 to 10 nm) are comparable
to the sizes of atoms, which makes them suitable for probing
the molecular/atomic structure of various materials. High-
energy X-rays penetrate deep into the samples; hence, they can
provide information on the structures of both the surface and
the bulk material. There are two most popular X-ray techniques
that can be used to characterize structural and surface proper-
ties of nanomaterials: X-ray photoelectron spectroscopy and
X-ray diraction.
XPS is the most widely used method for surface analysis of
nanomaterials and nanostructures,126,127 which is commonly
used for quantitative estimation of the surface composition,
lm thickness and surface sensitivity.4,119
XRD is a potent, contactless, and nondestructive technique
for identifying crystalline phases and determining the struc-
tural properties of nanomaterials, including the strain state,
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Nanoscale
grain size, epitaxy, phase composition, preferred orientation
and defective structures. XRD sensitivity strongly depends on
the object studied; the technique is the most sensitive for high-Z
structural elements.129,130
4.1 X-ray photoelectron spectroscopy or electron
spectroscopy for chemical analysis (ESCA)
The spatial resolution of XPS is lower than that of modern
leading-edge technologies; however, XPS remains an extremely
potent tool for obtaining the functional and structural data on
highly complex heterostructural systems.130132
Although XPS microprobes employing ion etching provide a
depth resolution on the level of 1 nm,133,134 their best lateral
resolution is only on the micrometric scale because of inherent
limitations of X-ray focusing.
XPS is used for characterization of a wide range of nano-
structured materials, including nanolayers,135 thin corrosion
lms,136 copolymeric light-emitting diodes,137 block polymer
lms,138 as well as nanoparticles. Also, XPS allows both nano-
particle size and electrical characteristics of coreshell nano-
particles to be evaluated.139
XPS can be used to determine the elemental composition of
the surface, resolve signature peaks of specic organic moieties
and, hence, dierentiate molecules on the sample surface.140
The XPS method has been used to monitor the reaction of
substituting pyridine for tri-n-octylphosphine oxide (TOPO) in
the layer covering the surface of nanocrystals, which is accom-
panied by redistribution of the electronic density of the crys-
talline substrate141 and changes in the surface state of TOPO-
capped nanocrystals.142 XPS has also been used for direct
demonstration of a uniform layer-by-layer growth of the CdS
shells of CdSe/CdS coreshell nanocrystals.143 In this case, the
change in the number of crystalline shell monolayers up to four
is observed by changes in the XPS signals.
Accurate evaluation of depth distributions of atoms and
nanostructures is crucial for nanotechnological applications.
Tougaard et al.144 have developed an XPS method for 3D
mapping of the upper 10 nm layer of specimens. X-ray photo-
electron spectra have been recorded to measure the thickness
(less than 10 nm) of the electrodeposited polyaniline coating
over helical carbon nanotubes.140
One of the tasks performed by using XPS is to check the
sample for heterogeneity, either lateral (in the plane of, or
parallel to the surface) or vertical (along the line perpendicular
to the surface). Lateral heterogeneities are detected by XPS
imaging, and vertical ones can be found by analyzing the energy
spectra at energies lower than the major peaks or deduced from
the dependence of the peak intensity on the emission angle.145
This is an important issue because the assumption on a
homogeneous depth distribution, which is oen made in XPS
surface analysis, may cause considerable errors. Most nano-
sized samples examined by XPS methods are actually hetero-
geneous in the direction perpendicular to the surface; however,
the data on the XPS peak intensity do not carry reliable infor-
mation on the depth distribution of atoms. This is illustrated in
Fig. 4, where conventional XPS analysis of the surface of gold
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Nanoscale
samples (ad) with dierent spatial distributions of copper
demonstrates the same peak intensities, showing the same
amount of copper on the upper surface, which is not the case.
The assumption that the depth distribution of Cu atoms is
homogeneous in all these cases and the surface concentration
being proportional to the peak intensity would result in an
inaccurate quantication. Hence, the peak intensity would
actually carry little information on the amount of Cu in the
surface layer, if at all.146
A combination of XPS analysis with the other methods is
oen used.147,148 For example, the location of the crystal phase
can be probed by comparing the ratios of the XPS and Auger line
intensities for the same atoms in bare and coreshell nano-
particles. This approach has been used to trace the growth of
ZnS on the surface of the CdSe nanocrystals.
Various XPS surface imaging tools are increasingly used for
analyzing dierent characteristics of biological systems,90,149 such
as the surface chemistry of microorganisms (by measuring the
peak intensities corresponding to dierent functional groups),150
formation of biolms,151,152 and for design of biocompatible
materials153 and pharmaceutical substances.154 Various aspects of
XPS analysis of dierent types of the samples and limitations of
this method are thoroughly reviewed in ref. 139.
There are extensive XPS databases facilitating the use of this
technique. For example, the United States National Institute of
Standards and Technology (NIST) has developed the Electron
Eective-Attenuation-Length Database155 and the database for
Simulation of Electron Spectra for Surface Analysis (SESSA).156
Fig. 4 Four signicantly dierent surface structures of copper atoms in gold that
give identical XPS intensities. Copyright 1996, AVS.146
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4.2 X-ray diraction
The so-called powder XRD is the most widely used XRD tech-
nique.130,157 Originally intended for probing powdery samples, it
has also become a powerful tool for analyzing liquid-suspended
particles,158,159 membranes160 and bulk polycrystalline solids.161
Powder XRD is suitable for both inorganic materials (e.g.,
metals and ceramics) and organic compounds (including
pharmaceuticals).162164
Powder XRD is used routinely when metal and metal oxide
nanoparticles are synthesized in a powder form to determine
whether these particles are crystalline or amorphous. Therefore,
it is important to determine the dominant crystalline phase in
the material as the thermodynamically most stable phase for a
given material may not be the same on the nanoscale.
XRD crystallography is the standard approach to solving
crystal structures. Single-crystal XRD is the optimal tool for
determining the geometry of molecular solids and individual
molecules, including the positions of atoms, the lengths (and,
hence, orders) of chemical bonds, the angles between them,
the shapes of coordination polyhedra, the molecule confor-
mations and the patterns of intermolecular contacts. The
technique allows the dierentiation between conguration
(cis/trans) isomers and, in many cases, optical isomers or
enantiomers. Moreover, modern XRD methodologies oer the
possibility of mapping the entire electron density pattern of a
crystal with the visualized electron clouds and direct deter-
mination of electric charges of specic atoms, electrostatic
potentials, etc.
The XRD technique is widely used in biological studies
today. The structures of large biological objects, such as
proteins, DNA complexes and virus particles, have been solved
by XRD.165167
XRD data have shown that very small particles (below 5 nm)
have unusual structural disorder that can substantially modify
the properties of these nanoparticles.168,169 Analysis of ca. 3 nm
ZnS particles showed that the strength of surfaceligand inter-
actions impacted the crystallinity of the interior of the nano-
particles. For smaller nanoparticles, surfaceligand interactions
are quite important and in a recent high resolution X-ray study
the structure of a thiol monolayer-protected gold nanoparticle
revealed a highly unusual structure of the surface functional
groups interacting strongly with each other and with the rst
layer of gold atoms.170
High-resolution powder XRD ensures sucient accuracy for
precision lattice constant determination and, hence, the eval-
uation of the structural and residual stresses in materials.171,172
Texture measurements of polycrystalline samples by
means of XRD are used to estimate the orientational distri-
bution of grains173 and dislocation densities174 in the sample.
The textured state is a transitional state between completely
random (powder) and completely oriented (single crystal)
states.175 XRD is also suitable for analysis of complex
crystalline structures such as the ve-fold coreshell nano-
crystals CdS/CdSe/CdS/CdSe/CdS. Their XRD pattern has
been found to be close to that of the cubic structure of CdS
crystals.176
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Time-resolved XRD has long been used to monitor solid-state 5 Optical spectroscopy techniques
reactions. The development of new radiation sources has made
5.1 Steady-state optical absorption and uorescence
it possible to trace phase transitions and physical changes over
spectroscopies
very short periods of time in the course of a reaction.177
The set of XRD techniques examining thin lms growing on In contrast to other methods optical spectroscopy requires no
substrates is termed thin-lm diraction. Its practical impor- physical separation process and provides quick results of high
tance is accounted for by implications of high-quality epitaxial quality in their specic application niches.
lms for microelectronics and optoelectronics.178 In general, studies on the nanoobject morphology do not
The use of synchrotron radiation in XRD is a recent inno- employ optical absorption spectroscopy. One of the rare
vation intended for local analyses in bulk polymers and exceptions is the evaluation of the mean sizes of quantum dots.
biopolymers. Wide angle X-ray scattering (WAXS)179,180 and These are mostly spherical nanoparticles of direct bandgap
small angle X-ray scattering (SAXS)130,180 are applicable to a wide semiconductors (CdSe, InP, PbS, etc.), in which the spectral
range of object sizes, beginning from interatomic distances. position of excitonic optical transition strictly depends on the
These techniques are suitable for analyzing samples in air, particle size unless it exceeds a certain limit (the exciton Bohr
matrices and environment cells. radius) due to the quantum-size eect (Fig. 5).180
Synchrotron radiation-based XRD analysis of multilayered The precise correspondence between the optical transition
semiconductors with layer thicknesses varying between 0.5 and energy and the size has been established for a number of
500 nm at a high spatial resolution is used in both scientic quantum dots, including CdSe, CdS, CdTe, ZnSe, PbS, PbSe and
research and technological developments.161 Advances in InP.190193 The steady-state optical absorption spectrum of a
focusing/imaging optics and detector technologies provide monodisperse ensemble of quantum dots comprises a series of
deep insight into the understanding of various phenomena well-resolved exciton peaks. Therefore, the mean quantum dot
pertaining to cell and molecular biology,181,182 as well as semi- size can be quickly determined from the position of the rst
conductor physics.165 For example, combined XRD and TEM excitonic transition peak with an accuracy of about 1020%.194196
approaches have yielded evidence that coreshell nanocrystals
are actually single crystals. Imaging of the patterns of crystal
lattice defects has shown that they remain uninterruptible
across a nanocrystal, which indicates epitaxial growth.143
In SAXS measurements, typical scattering angles are smaller
than one degree.130 According to Bragg's law, this is the range
where the diraction parameters convey information about
structures with long d-spacing. Therefore, SAXS is generally
applied to thin lms183 and biological macromolecules, such as
proteins and nucleic acids.184,185
Extended X-ray absorption ne structure (EXAFS). The
EXAFS technique deals with X-ray absorption in the range
slightly higher than the absorption limit of the element in
question. EXAFS results from interference of the spectrum of
photoelectrons ejected from irradiated atoms to that of back-
scattered electrons reected by neighboring atoms. EXAFS
provides structural information in a 6 A vicinity of the atom in
question at room temperature and at longer distances at lower
temperatures. This technique is used for highly accurate
measurement of interatomic distances. Thin oxide lms,186
binary alloyed nanocrystals187 and ternary quantum dots91 are
common objects of XAFS analysis.
The abundant results of X-ray crystal structure analyses are
available in a number of international databases. The structures
of small organic and organometallic molecules can be found
in the Cambridge Structural Database (CSD; Cambridge, UK),
a large source of molecular metric data (http://
www.ccdc.cam.ac.uk).
The Crystallography Open Database188 contains information
on the structures of organic, inorganic and organometallic
substances.
Powder diraction data for more than 600 000 substances Fig. 5 Observation of discrete electronic transitions in optical absorption is a
are available from the International Centre for Diraction Data measure of the wealth of spectroscopic information that can be uncovered in
(ICDD).189 monodisperse nanocrystal samples (s # 5%). Copyright 2000, Annual Reviews.180

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Nanoscale
Non-spherical (e.g., nanorods) and composite nanoparticles
(e.g., coreshell quantum dots) represent a more complicated
case.197202 It is known that for many AIIBVI quantum dots
emitting uorescence in the visible range (e.g., CdSe, CdS, CdTe
and ZnSe),203,204 the mean size can also be evaluated from the
spectral position of the uorescence emission band.
The absorbance spectra of plasmonic nanoparticles (e.g.,
gold and silver) also reect their morphometric characteristics.
Their high absorbance in the visible region results from exci-
tation of surface plasmons,205 the energy of which depends on
the nanoparticle size and shape. Specically, the plasmonic
absorption band of spherical silver particles is shied to the
low-energy range if their sizes exceed 3040 nm.206 In the case of
ellipsoid nanoparticles, there are two plasmonic peaks (those of
the transverse and longitudinal plasmons), the spectral posi-
tions of which can be used to determine the relative lengths of
the two axes of the ellipsoid.206,207 However, the morphology
absorbance relationship in plasmonic nanoparticles is too
complex for practical use.207209 The dynamic light scattering
methodology is typically used in this case, especially in bio-
logical research.210
5.2 Dynamic light scattering or photon correlation
spectroscopy
Dynamic light scattering (DLS) is the most suitable technique
for estimation of the sizes of nanoparticles dispersed or dis-
solved in solvents.211 The dimension obtained using DLS is
called the hydrodynamic radius, since it describes the radius of
a particle moving as a unity in a uid, together with a thin
tethered layer of the surrounding medium.212 The technique is
applicable to a wide range of particle sizes, from several nano-
meters to several micrometers (Fig. 6).
Fig. 6 Schematic presentation of particle size distribution in the case where 50% of the particles are 5 nm in diameter and 50% of their agglomerates are 50 nm in
diameter. (a) Distribution of the number of particles; (b) distribution of their volume or weight; (c) distribution of the intensity of light scattered by them. Copyright
2004, Malvern.220
8792 | Nanoscale, 2013, 5, 87818798
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DLS is one of the most widely used methods for character-
ization of complex liquids, such as colloidal suspensions,213
polymer solutions214 and gels.215 DLS provides a measure of the
time scale for uctuations in the index of refraction of complex
liquids. However, conventional DLS has its inherent limita-
tions. The sample concentration must be high enough to ensure
an adequate signal, but the risk of erroneous results due to
multiple scattering (light scattered by one particle undergoing
scattering by another) applies specic restrictions on the
concentration range for which measurements are valid. The
multiple scattering eect in DLS experiments can be overcome
by using the cross-correlation procedure.216 This technique
relies on the cross-correlation of two measurements to extract
single-scattering information from the same scattering volume
and the same nominal scattering vector. Regarding the sizes of
suspended particles, one should take into account the thickness
of the liquid layer on the particle surface that moves along with
it.217 The particle size in a suspension is a crucial characteristic
of specic products, and it is routinely monitored together with
the zeta-potential.218,219
DLS has been used to estimate the hydrodynamic diameters
of CdSe nanocrystals and cluster molecules, including their
ligand shells, ranging in size from 1 to 10 nm.221,222 All these
studies, however, have been performed in homogeneous media
that ensured optimal conditions for the measurements. Char-
acterization of nanoparticles in more complex liquid media or
matrices, such as biological tissues, faces considerable prob-
lems. First, the type of the medium should be taken into
account, in particular, one should know whether this is a pure
homogeneous medium or a complex heterogeneous system.
Second, various conditions in vivo may aect the surface
composition and aggregation behavior of nanoparticles.
Biomolecules may be adsorbed on them and substantially alter
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their hydrophobicity, charge and charge distribution. The
adsorbed biomolecules form a biomolecule corona around
nanoparticles, giving them biological identity.223
DLS is also applied to the study of proteins and other
biomolecules. In this case, processes such as aggregation,
nucleation and denaturation are readily detectable.224
Like DLS, the uorescence correlation spectroscopy (FCS)
technique extracts information on particles or molecules from
their Brownian motion. The dierence is that FCS deals with the
uorescence of the objects rather than light scattering on them
and involves correlation analysis of the uorescence intensity
uctuations.224227
FCS allows uorescence-labeled molecules to be observed in
living cells, thereby paving the way to in situ or in vivo
biochemistry.228230
Dual-color cross-correlation FCS has been used to trace DNA
amplication during PCR; for this purpose, the authors used
primers tagged with dierent uorophores.231 This procedure
may provide a reliable, convenient and highly sensitive method
for detecting viruses; it has already been used to evaluate the
titer of HCV in blood serum.232
The engineering of cells containing genetically tagged
proteins (such as green uorescent protein) oers the possi-
bility of studying molecular dynamics in vivo with the use of
FCS.233,234
6 Summary and outlook
Progress in nanotechnology is closely related to the develop-
ment of nanometrology. The number of novel nanomaterials, as
well as the areas of their applications, is constantly growing,
which requires comprehensive analysis of their morphological,
chemical, electromagnetic and other parameters. This calls for
further development of the existing measurement techniques
and designing of new ones. The methods available to date allow
researchers to obtain information on both individual molecules
or nanoparticles and their ensembles.
When handling nanomaterials, one should bear in mind the
ne structure of nanosized objects, taking into consideration
the properties entailed by their sizes and their sensitivity to
external factors. Underestimation of their specic characteris-
tics may considerably bias the results.
Thus, precise evaluation of various parameters of objects
smaller than 100 nm and adjustment of the existing techniques
to the specicity of each particular type of material are key
problems of nanometrology.
There is a specic set of methods for analysis of each type of
nanomaterial. Two electron microscopic techniques, TEM and
SEM, provide information on the internal structure of a spec-
imen and its surface, respectively. The numerous additional
modes of these techniques allow the characteristics of the
specimen to be explored in more detail. The use of scanning
probe microscopy techniques makes it possible to examine a
surface with a precision of up to one atom, readily yields 3D
images on the nanometer level and allows manipulation of
single atoms or molecules. X-ray methods are mainly used to
obtain precise information on the internal structure of objects.
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Optical methods are highly specialized but ensure quick and
accurate measurement of nanosized objects in their respective
application areas.
The diversity of methods outlined here oers numerous
possibilities for studying nanomaterials. We hope that this
review will help the readers who are new to the eld to choose
the optimal techniques for their studies.
Acknowledgements
This study was supported by the Ministry of Education and
Science of the Russian Federation (grant no. 11.G34.31.0050
and 14.512.11.0034), the European Commission through the
FP7 Cooperation Program (grant no. NMP-2009-4.0-3-246479),
and the programs HYNNOV and Nano'Mat supported by the
Champagne-Ardenne region, the DRRT Champagne-Ardenne
and the FEDER. We thank Vladimir Ushakov for his help in the
preparation of the manuscript.
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