Nanotoxicology, 2010; Early Online, 1–12

Cellular uptake and intracellular fate of engineered nanoparticles: A

review on the application of imaging techniques

RATNA TANTRA & ALEX KNIGHT

National Physical Laboratory, Teddington, Middlesex, UK

(Received 4 February 2010; accepted 19 July 2010)
Nanotoxicology Downloaded from informahealthcare.com by Charing Cross & Westminster Medical Sch on 01/14/11

Abstract
The use of imaging tools to probe nanoparticle-cell interactions will be crucial to elucidating the mechanisms of nanoparticle-
induced toxicity. Of particular interest are mechanisms associated with cell penetration, translocation and subsequent
accumulation inside the cell, or in cellular compartments. The objective of the present paper is to review imaging techniques
that have been previously used in order to assess such interactions, and new techniques with the potential to be useful in this
area. In order to identify the most suitable techniques, they were evaluated and matched against a list of evaluation criteria. We
conclude that limitations exist with all of the techniques and the ultimate choice will thus depend on the needs of end users, and
their particular application. The state-of-the-art techniques appear to have the least limitations, despite the fact that they are not
so well established and still far from being routine. For example, super-resolution microscopy techniques appear to have many
advantages for understanding the details of the interactions between nanoparticles and cells. Future research should
concentrate on further developing or improving such novel techniques, to include the development of standardized methods
and appropriate reference materials.

Keywords: Nanoparticles, cellular imaging, nanotoxicity, in vitro

For personal use only.

Introduction represents possible pathways for nanoparticle inter-

The recent dramatic growth in nanotechnology
research and applications has resulted in widespread
concern about the potential toxicity of nanoparticles.
This concern arises because they may possess differ-
ent properties to the corresponding bulk materials,
which could give rise to harmful effects not detected
by conventional toxicological approaches. In partic-
ular, it is the extremely small size and large surface
area to mass ratio that has often been linked with
pathogenicity (Warheit et al. 2009). Many aspects of
nanoparticle behaviour must be understood in order
to determine their toxicity and elucidate the mechan-
isms. These will include studies at the whole organism
level, in order, for example, to understand routes of
entry to the body and of clearance and excretion from
it; and studies of the surface binding of biomolecules,
which is likely to strongly mediate their effects in vivo.
However, one of the major concerns is the capacity of
nanoparticles to penetrate cells and their subsequent
ability to accumulate in specific sub-cellular orga-
nelles, such as the nucleus; Figure 1 schematically
actions with cells.
Clearly there is a need for better understanding of
how nanoparticles can pass through the cell mem-
brane, and their subsequent interaction with the cell’s
internal structure. Hence, the ability to visualise local
populations of nanoparticles at high resolution within
cells is important in order to deduce the mechanisms
of any toxicity (Salonen et al. 2008).
In the past, imaging-based tools have been dem-
onstrated to be valuable in the investigation of
nanoparticle-cell interactions, particularly in asses-
sing their location and distribution within cells. As
nanotoxicological research is increasingly dependent
on such tools, technologies are continuously being
developed to improve their performance in probing
nanoparticle-cell interactions. The main purpose of
this article is to present a review of imaging techniques
and of their utility to study nanoparticle-cell interac-
tions, with the aim of elucidating the mechanism of
toxicity at a cellular level. Much of the source material
presented for this paper is from peer-reviewed papers,
with relevance to the objectives stated. This review has

Correspondence: Dr Ratna Tantra, National Physical Laboratory, Hampton Road, Teddington, Middlessex, TW11 0LW, UK. E-mail: ratna.tantra@npl.co.uk.

ISSN 1743-5390 print/ISSN 1743-5404 online
2010 Informa UK, Ltd.

DOI: 10.3109/17435390.2010.512987
 2 R. Tantra & A. Knight
3 1
9 given instrumentation to study nanoparticle-cell
? interactions will be determined mainly by the needs
2 of the toxicologists. At present, such criteria are
4 although considered to be highly subjective in nature,
6 to be taken as a starting point for the evaluation of the
?
7
5 follows:
Nanotoxicology Downloaded from informahealthcare.com by Charing Cross & Westminster Medical Sch on 01/14/11
?
8
Figure 1. Possible interactions of nanoparticles with cells. This
Figure schematically represents possible pathways for nanoparticle
interactions with cells. Not all of these processes have been docu-
mented. Extracellular nanoparticles (1), represented by diamonds,
may bind proteins and other biomolecules (2), e.g., albumin, which
may modulate their interactions with cells. Nanoparticles may be
taken up by endocytosis (3) or pass directly through the membrane
(4) or simply interact with the membrane surface (5). Endocytosed
nanoparticles will be enclosed in membrane-bound organelles, but
For personal use only.
may be able to escape into the cytosol (6), or alternatively may
accumulate in endosomes or lysosomes, where degradation may
occur (7). Alternatively, there may be pathways, such as exocytosis,
by which nanoparticles are expelled from cells (8). Nanoparticles
may also be transported to, or accumulate in, specific organelles
such as the nucleus (9).
been divided into several parts. The first section will
identify and provide set of evaluation criteria, which
aims to assist in identifying those tools considered to
be ‘fit for purpose’. The second section presents
potentially suitable techniques (both well established
and ‘state-of-the-art’), for such a purpose. For each
technique, technical background information, pros/
cons and their applications for studying nanoparticle-
cell interactions in the literature, will be presented.
This will be followed by a summary section that aims
to evaluate and compare the different techniques,
benchmarking them against criteria identified; issues
surrounding the current state of standards/standard-
ization will also be addressed. It is not the intent of
this paper to delve into the details of the individual
techniques but rather to give an overview, and to
provide sufficient understanding to identify the
most suitable imaging technique for the purpose of
probing nanoparticle-cell interactions in nanotoxico-
logical investigations.
Criteria used to evaluate the appropriateness of
the imaging tools to study nanoparticle-cell
interaction
The required criteria for the appropriateness of a
ambiguous and have not been well established or
evaluated. We have thus proposed a set of criteria,
different imaging tools. The criteria identified are as
(a) High selectivity (or contrast) for nanoparticles.
The instrument should be able to effectively
distinguish the nanoparticles from the back-
ground; there should be sufficient selectivity to
not only locate the nanoparticles inside the cells
but also within cell compartments.
(b) High sensitivity. The instrument should be able
to detect and monitor the fate of nanoparticles
within cell, with sufficient sensitivity (ideally) to
detect at single nanoparticle level. The ability to
detect single isolated nanoparticles is of huge
importance as nanotoxicological activity is very
much dominated by size (Limbach et al. 2005).
(c) High resolution. Ideally it should be possible to
distinguish individual nanoparticles from aggre-
gates thereof and to localise them precisely
within the cell.
(d) Imaging of cell structure. The method should
provide information on the structure of the
cell so that the location of nanoparticles can
be determined. For example, the ability to
visualise organelles allows sites of nanoparticle
accumulation to be determined. Alternatively,
markers for cellular responses to nanoparticles
can be visualized. This will typically require
some form of multiplex detection.
(e) Imaging of whole and sectioned cells. Ideally, the
imaging technique should be able conduct sur-
face analysis of the cell structures as well imag-
ing the fate and subsequent localization of the
nanoparticles once they have internalized by the
cells.
(f) Label free detection. This is the ideal, as the use of
labels (such as fluorescent tags or radioactive
probes) can result in the incorrect conclusions
being drawn from the data; if labels are used then
mechanism behind the toxicity will be based on
the properties of the nanoparticle-label complex
rather than the inherent properties of the nano-
particles themselves.

(g) Non-invasive methods. Where possible, non-
destructive methods (including protocols asso-
ciated with sample preparation, as well as
analysis) are very much needed. Methods that
are less disruptive to the cells are favoured. The
ability to conduct live-cell imaging is ideal, as it is
important for toxicologists to understand the
mechanism of toxicity associated with living
processes.
(h) Routine/accessibility. The technique should have
the ability to conduct the analysis with high
accuracy, reproducibility and high throughput.
This is important if ‘endpoints’ for routine
toxicity studies using imaging tools are to be
Nanotoxicology Downloaded from informahealthcare.com by Charing Cross & Westminster Medical Sch on 01/14/11
developed and for the effective interaction with
regulatory agencies to be subsequently estab-
lished. Ideally, the protocols associated with rou-
tine analysis surrounding sample preparation and
analysis should be well established and past stud-
ies have shown the feasibility of using such tools
to study nanoparticle-cell interactions.
(i) Yield complementary chemical information. This is
important in order to identify ‘suspect’ nanopar-
ticles; ‘suspect’ nanoparticles are those nanopar-
ticles that have a different chemical make-up
to those under study and thus can poten-
tially interfere with the toxicological response
For personal use only.
of nanoparticles.
Imaging tools
Light microscopy
Light microscopy is widely used in the life sciences for
cell imaging. Among the advantages of light micros-
copy is the capability to image living cells, or fixed
cells with minimal sample preparation. A variety of
contrast mechanisms are available, including label-
free modalities such as phase contrast and differential
interference contrast (DIC). However, in many cases,
the most powerful forms of light microscopy are those
based around fluorescence.
In all forms of light microscopy, there is a
fundamental limit on the resolution that is obtained
(about half the wavelength of the light used), because
of diffraction. Whilst in some cases this limit can
be reduced or by-passed using special techniques
(discussed below), it is almost always the case that
individual nanoparticles will be too small to be
resolved. However, this does not mean that light
microscopy is unsuitable for imaging studies of nano-
particles. For example, nanoparticles may still be
imaged by fluorescence even if they cannot be
resolved (appearing like a point source). This may
Review of imaging techniques 3
be sufficient for the purpose of localizing the nano-
particles within a cell. Furthermore, aggregates of
nanoparticles may be sufficiently large to be resolved.
A predominant issue in the use of light microscopy
is whether the available contrast mechanisms are
suitable for the nanoparticles of interest. Bright-
field microscopy, relying on the absorption of light,
or phase contrast microscopy, relying on a difference
in refractive index, are unlikely to provide sufficient
contrast because most nanoparticles will be too small,
or too low in mass, to be detected. For highly scat-
tering nanoparticles, dark-field microscopy or reflec-
tion microscopy may be suitable. Fluorescence is one
of the most powerful contrast mechanisms available in
light microscopy, because of its great sensitivity, cou-
pled with the range of wavelengths that can be used to
follow multiple labels in one sample. Thus in one
experiment, the nanoparticles themselves, together
with markers for cellular structures or responses,
can be imaged simultaneously. Some classes of nano-
particles, such as quantum dots, are intrinsically fluo-
rescent and this makes them ideal candidates for this
type of study. Other classes of nanoparticles may
be derivatized with fluorescent markers such as
Fluorescein-5-isothiocyanate (FITC).
The most common forms of fluorescence micros-
copy are epifluorescence and confocal laser scanning
microscopy. In epifluorescence, typically an arc lamp
is used to illuminate the field of view, with filters used
to select the excitation and emission wavelengths
of the probe of interest. Because of this wide-
field illumination and detection, fluorescence from
out-of-focus objects is superimposed on that from
objects that are in focus. This reduces the contrast
and resolution of the technique. In confocal laser
scanning microscopy, the fluorescence is excited
using a laser beam. The laser is focused to a point
that is rastered across the field of view. Although there
are many variations, typically both the laser and the
emitted fluorescence pass through a pinhole in the
focal plane of the objective. This pinhole removes
the out-of-focus fluorescence, improving contrast and
resolution and also providing the ability to optically
‘section’ the sample. Multiple such sections or sli-
ces can be built up, enabling the three-dimensional
reconstruction of samples.
Confocal microscopy has been shown to be partic-
ularly valuable to probe cell-nanoparticle interactions,
in particular to show uptake of nanoparticles into
the cells (Calvo et al. 1994; Gorelik et al. 2002;
Dailey et al. 2003; Liu et al. 2004; Kemp et al.
2008). In particular, Salonen et al. (2008) have
used this imaging tool, to reveal the uptake of
carbon-based nanomaterials into cells, which subse-
quently resulted in contraction of cell membranes; the
 4 R. Tantra & A. Knight

contraction effect was attributed to the aggregation of
nanoparticles promoted by cell surfaces. In order to
investigate the mechanism of toxicity, such techni-
ques are often employed in conjunction with other
techniques giving complementary information, or
possibly using multiple fluorescent probes. Again,
past studies have indicated that surface characteristics
of the nanoparticles play a dominant role in the
mechanism of toxicity (Gupta and Curtis 2004;
de la Fuente et al. 2007).
Despite such advantages, fluorescence imaging has
the following limitations:
(a) In general, a much reduced sensitivity and spatial
Nanotoxicology Downloaded from informahealthcare.com by Charing Cross & Westminster Medical Sch on 01/14/11
state of the fluorescent group. This approach has the
advantage that this lifetime is very sensitive to the
nature of the fluorescent group and its environment,
which may help to reduce background. It also has the
added advantage of minimising the effect of photon
scattering in thick layers. Hence, such an ‘add-on’
tool will be particularly useful for probing greater
depths in thick samples (Lakowicz 1994). For exam-
ple, Conroy et al. (2008) have used FLIM to suc-
cessfully image the accumulation of quantum dots in
the nuclei and nucleoli of living human cells.
Although the confocal optics scheme ensures that
photons are collected mainly from the focal plane
(so-called ‘optical sectioning’), it is possible to

resolution compared to electron-based imag-

improve this sectioning process further. In multi-
ing techniques. Although single molecule fluo-
photon excitation (MPE) fluorescence, two or more
rescence has long been possible, this is very
photons must be absorbed simultaneously to excite
much dependent on both the nature of the
fluorescence. For example, instead of one photon at
fluorescing species and detection capability of
532 nm, two photons at 1064 nm might be required.
the instrument; single molecule detection is real-
This approach has a number of advantages. Firstly,
ized by having appropriate combination of the
this is a non-linear process, which means that the
fluorescing species being extremely bright and
excitation volume is much smaller than for confocal
the detector sufficiently sensitive and elimination
microscopy, reducing out-of-focus fluorescence still
of background fluorescence (Su et al. 2004;
further and reducing photodamage. Secondly, the
Michalet et al. 2007).
longer wavelength photons are scattered less by
(b) Potential need for labelling. As previously men-
biological tissue, and therefore this technique is
tioned, unless the nanoparticles are inherently
ideally suited for imaging thicker samples than can
For personal use only.

fluorescent, it is normal procedure to prepare the

be tackled by conventional confocal microscopy
samples by targeting a fluorescent probe to the
(Halbhuber and Konig 2003; Dufour et al. 2006).
nanoparticles. However, attaching such probes
In the past, workers have shown the potential of
will affect the interpretation of the data, as such a
such a tool, by using quantum dots in order to
fluorescent marker will undoubtedly change the
observe detailed cell structures (Ferrara et al.
physico-chemical make-up of the nanoparticles
2006). Although a relatively novel technique, efforts
and any observed toxicity may be attributable to
have been made by Glaschick et al. (2007) to image
the marker rather than the nanoparticles; it is
nanoparticle-cell interaction.
the nanoparticle-label complex rather than the
As noted previously, a significant limitation of
inherent properties of the nanoparticles that will
optical approaches is their diffraction-limited resolu-
elicit the cell response.
tion. However, recent years have seen the develop-
(c) Potential sample damage. The process of fluo-
ment of a number of microscopy techniques which
rescence excitation can itself be harmful, in that
cleverly by-pass this barrier (Hell 2008). Since dif-
fluorescent molecules can be harmful in an
fraction cannot be avoided, these approaches use a
excited state or the interaction of light with the
variety of different tricks to ‘switch off’ a subset of
cell itself can result in phototoxicity. For exam-
molecules, so that their locations can be imaged
ple, it is known that UV or blue light used to
consecutively. The most common methods rely on
excited fluorescence may also interact directly
stimulated emission, photoswitchable fluorophores,
with DNA or other cell components, to ulti-
or structured illumination, and sometimes combina-
mately induce toxic damage (Matsumoto and
tions thereof. These techniques have demonstrated
Wilson 2002). The extent of damage will
the possibility of imaging cellular structures at resolu-
undoubtedly be enhanced if under conditions
tions as low as 10 nm (Hell 2008). Typically they use
of prolonged exposure to the light source.
‘photoswitchable’ small-molecule fluorophores for
A number of variants of fluorescence imaging exist, imaging, but recently, direct super-resolution imaging
although only a few have been employed to probe cell- of quantum dots has been demonstrated (Fernández-
nanoparticle interactions. FLIM (Fluorescence Life- Suárez and Ting 2008; Irvine et al. 2008). This opens
time Imaging Microscopy) is a tool that is used to the way to the use of super-resolution optical imaging
produce an image based on the lifetime of the excited of nanoparticle interaction with cells.

Scanning probe microscopy
The set of techniques under the ‘scanning probe
microscopy’ umbrella, relies on the use of a physical
probe (sometimes referred to as the tip) to raster
across a specimen; it is the interaction between the
probe and the surface of the specimen (as a function
of position) that is monitored, in order to produce
an image on the basis of theoretical/experimental
assumptions of such interactions. The way image
obtained in this way can be seen as ‘indirect’ in
nature, as it uses a probe to ‘see’, much in the
same way as ‘a blind person reading Braille’. Cur-
rently, there are over 20 well-established SPM
Nanotoxicology Downloaded from informahealthcare.com by Charing Cross & Westminster Medical Sch on 01/14/11
techniques, but unlike the electron and confocal fluo-
rescence microscopy the application of such techni-
ques to study cell-nanoparticle interactions are very
much limited (Wiesendanger 1998).
In the field of cellular imaging, Atomic Force
Microscopy (AFM) is considered to be one of the
most commonly used tools (Li 2005). In AFM, the
probe is a fine cantilever and it is the ‘interaction
force’ between the tip of the probe and surface that is
measured, to form a high-resolution image. The
image will thus be dependent on the interaction force
between the sample and probe tip and the distance
between them. Past studies have used AFM as a tool,
For personal use only.
not on its own, but to further complement other
techniques that offer direct imaging. For example,
past workers have found AFM to be particularly
useful to quantify the force of nanoparticle-cell mem-
brane interactions (Leroueil et al. 2007; Vasir and
Labhasetwar 2008). In addition, Leonenko et al.
(2007) have used the AFM probe tip to ‘model’ the
nanoparticle itself; it is the forces of interaction
between the tip and the cell that is measured, so as
to study the adhesive interaction between nanoparti-
cles and lung epithelial type 2 cells.
As a tool, AFM has many advantages, to include the
ability: To offer high spatial resolution (fractions of a
nanometre), to apply forces as low as 20 pN and to
measure in liquid environment. However, AFM has
several disadvantages, to include its restrictions
concerning:
(a) Image size, in which it is capable of imaging with
maximum height on the order of ~ mm, with
maximum scanning area of ~ 150  150 mm;
(b) Quality of image, which will be limited by the
radius of curvature of the probe tip. An incorrect
choice of tip for the required resolution can thus
lead to image artefacts;
(c) Limitations to be used as routine tool. The
technique is not capable of real time scanning,
often requiring several minutes for a typical scan;
Review of imaging techniques 5
(d) Possible artefacts. AFM images are affected by
hysteresis of the piezoelectric material and cross
talk between the x, y and z axes. Thus software
enhancement and filtering are often required in
order to achieve meaningful results. In addition,
the slow rate of scanning can lead to thermal
drift;
(e) It cannot detect or measure nanoparticles inside
a living cell.
Recently, the AFM technique has been developed
further to enhance its measurement capabilities,
so as to make the platform more suitable to effec-
tively probe nanoparticle-cell interactions. This rev-
olutionary approach is known as Scanning Near Field
Ultrasound Holography (SNFUH); the measurement
involves launching a high frequency (~ 100s kHz to
several MHz) acoustic wave from the bottom of the
specimen, while another wave is launched on an AFM
cantilever, resulting in an ‘acoustic interference’. The
AFM tip thus acts as an antenna for the acoustic
interference in the near-field regime, offering a
‘quantitative’ account of internal microstructure of
the specimen. Although the platform is extremely
novel, the potential of such a technique is huge; the
main advantage is in its ability to image soft samples
and to probe structures that are well below the sur-
face. Hence, researchers do not need to cut up the cell
(as in electron microscopy) or inject light emitting
molecules (as in fluorescence microscopy) in order to
probe nanoparticles-cell interaction. Shekhawat and
co-workers have shown the viability of this technique
to probe cellular uptake of nanoparticles, in particular
to show the cellular fate of single-walled carbon nano-
horns within lavage cells and blood (Shekhawat and
Dravid 2005; Tetard et al. 2008).
Another variant to the traditional AFM also include
Scanning Ion Conductance Microscopy (SICM),
which have in the past been useful for the imaging
of live cells (Korchev et al. 1997, 2000, 2007;
Mann et al. 2002; Gorelik et al. 2004, 2008;
Shevchuik et al. 2006; Cognard et al. 2008). This
technique acquires topographic images by keeping
the ionic current constant instead of applying a physical
force to the sample and hence more ideal if compared
to conventional AFM (Edwards et al. 2009). SICM
however have not been fully investigated for the study
of live cell-nanoparticle interactions.
Electron-based imaging technologies
This technology is based on using a focused electron
beam to excite simultaneously a number of physical
processes (to be converted to corresponding electrical
signal variations) in order to form an image. This
 6 R. Tantra & A. Knight
complex interaction results in a highly resolved image,
which contains information about the spatial variation
across the specimen. Overall, such technology can
be mainly subdivided into two: Scanning elec-
tron microscopy (SEM) and Transmission electron
microscopy (TEM). These are well-established tech-
niques that have been used in the past to probe cell-
nanoparticle interaction.
In SEM, the focused electron beam is swept across
the sample, often resulting in the emission of second-
ary electrons and backscattered electrons, to produce
either the corresponding secondary electron image or
the backscattered electron image, respectively. To
extract valid information via SEM, it is necessary to
Nanotoxicology Downloaded from informahealthcare.com by Charing Cross & Westminster Medical Sch on 01/14/11
understand the various contrast mechanism under
different imaging conditions. If images are generated
using secondary electrons, then contrast will be
extremely sensitive to surface topography, in which
the number of electrons collected from a surface will
ultimately be governed by the variation in the angle
between the incident electron beam and the local
normal to the surface of the specimen. If an image
is generated using backscattered electrons, then image
contrast will be sensitive to atomic weight of dif-
ferent regions of the sample. In addition to these,
the following factors can affect image contrast: volt-
age/electric field contrast, magnetic contrast, beam-
For personal use only.
induced conductivity contrast, crystallographic
orientation contrast, transmission mode contrast,
cathodoluminescent mode contrast, temperature var-
iation contrast and contrast caused by specimen irra-
diation (Clarke 1970).
In TEM, the electron beam is transmitted through
an ultra-thin specimen and it is the unscattered,
transmitted electrons that are focused on onto an
imaging device. As the electron wave traverses the
specimen, it can change both in its amplitude and
phase. Two types of image contrast govern a TEM
image: Amplitude and phase contrast. Both types of
contrast can contribute to an image simultaneously
but one usually dominates. Amplitude contrast has
two principal types, namely, mass thickness contrast
and diffraction contrast; the former is the most critical
contrast mechanism for amorphous materials such
biological materials. Mass contrast arises from two
factors: Mass/atomic number of the element con-
tained in the specimen and the thickness of the
specimen. If thickness is homogeneous, then elec-
trons interact with heavy atoms stronger than with
light atoms; this explains the need to stain soft tissues/
cells with heavy materials, such that ultrastructures in
the specimen can be observed. If thickness is not
homogeneous, then thicker areas appear darker in
the resulting TEM image, as more electrons are
scattered in thick areas than in thin areas of the
specimen. Phase contrast, on the other hand, results
from the interference between electrons and subse-
quently the electrons will have different phases after
passing through the specimen. As a result, phase
contrast can be used to directly image the crystal
structure of a specimen at atomic resolution (Williams
and Carter 2009).
Both SEM and TEM have been employed in the
past to obtain internal structure of cells, but biggest
factor affecting quality is the need for extensive sam-
ple preparation. For SEM sample preparation, sample
preparation steps may include: drying (in a freeze
dryer or critical point dryer), attaching sample to
stub and coating with conducting material prior to
analysis. For TEM analysis, steps may include: fixing,
dehydrating, embedding, sectioning on ultramicro-
tome, mounting on grid and staining (often with
heavy metals) prior to analysis (Dykstra 1993).
Care is needed to ensure that these invasive processes
do not give rise to artefacts.
The high spatial resolution offered (~ 1 nm
and ~ 0.08 nm for high resolution SEM and TEM,
respectively) will mean that such instruments will not
only have the desired selectivity (with the ability to
differentiate nanoparticles against cell/cell compo-
nents) but are sufficiently sensitive to differentiate
isolated nanoparticles from the corresponding aggre-
gates. It is noteworthy to mention that secondary
electrons are normally used to generate an SEM
image based on topographic contrast; here, contrast
is thus based on detecting areas of different atomic
numbered elements (Z-contrast). If the specimen is of
relatively low atomic number then there is a need to
coat/stain with heavy metals (such as silver) in order to
increase the numbers of backscattered electrons in
order to improve on signal resolution and selectivity
(Hayat 2000).
Past workers have used these techniques, often in
combination with complementary techniques, to fur-
ther deduce the mechanisms of toxicity. From such
studies, workers have proposed that nanoparticle
parameters such as size and surface properties, will
govern steps associated with cellular binding, subse-
quent incorporation of nanoparticles into cells and
eventual cytoxicity (Gupta and Curtis 2004; de la
Fuente et al. 2007; Panessa-Warren 2008).
In the past, out of the two techniques, TEM is often
the tool of choice (due to the much higher spatial
resolution in TEM) to observe nanoparticle:
(a) Penetration through a cell membrane (Rothen-
Rutishauser et al. 2006; Verma et al. 2008);
(b) Translocation into cell compartments (Kaul and
Amiji 2005; Nativo et al. 2008).

(c) Despite the clear potential advantages of using
such state of the art methods, a number of
limitations remain:
(d) Potential sample damage and introduction of arte-
facts under imaging electron beam. This is partic-
ularly true if the sample has been exposed to the
beam for a long period (Pan et al. 2006).
(e) Unsuitable as methods for routine measurement.
Sample preparation is extremely laborious and
very much dependent on the skill of the operator,
in which cells have to be sectioned and fixed on
to substrate surfaces. Ultrathin sectioning to
achieve the desired sample thickness in TEM-
based methods are crucial, so as to allow the
Nanotoxicology Downloaded from informahealthcare.com by Charing Cross & Westminster Medical Sch on 01/14/11
electron beam to pass through the specimen
(Schwarz and Humbel 2008).
(f) Need to perform the analysis under high vacuum
conditions. Hence, the samples will be dead and
chemical fixation steps are necessary in order to
preserve the cell in as ‘near to life’ conditions as
possible.
(g) Potential need for staining. It is possible that sec-
tions on grids can imaged without heavy metal
staining (for example of uranyl acetate and lead
citrate); additional contrast enhancement can be
achieved with the utilization of smaller apert-
ure sizes with the objective lens of TEM and
For personal use only.
if images are obtained at low voltages, i.e.,
60–80 kV. However, the staining process allows
the experimentalist to better identify subcellular
structures and organelles, which will subse-
quently mean the ability to identify nanoparticles
within.
Recent developments of research activities in this
area include the use of TEM technology of variant
designs, to include the use of ‘state-of-the-art’ Scan-
ning Transmission Electron Microscopy (STEM)
and Energy-Filtered Transmission Electron Micros-
copy (EFTEM) (Rodenburg 1997). STEM, is a TEM
variant that makes use of an SEM-like beam, that is, it
rasters a fine beam across the sample, in order to
obtain high contrast images. EFTEM is another var-
iant of TEM that employs an energy filter to separate
the transmitted electrons according to their energy.
By doing so, only electrons of particular kinetic ener-
gies are used to form the image or diffraction pattern,
which improves the image contrast. Porter and co-
workers have shown that both STEM and EFTEM
techniques are capable of visualizing cell structures
(such as membranes, the mitochondria, ribosomes
and the nucleus) without the need for traditional
staining techniques. Such tools were employed to
study the uptake of C60 by cells, to result in
images showing the corresponding 3-dimensional
Review of imaging techniques 7
nanoparticle distribution within the cell (Porter
et al. 2006, 2007a). These techniques were shown
to be powerful enough (without the need for staining)
to image the interactions of single walled carbon
nanotubes with cells. Studies have shown that prior
to cell entry, nanotubes were able to fuse with cell
membrane; this is followed by penetration and local-
ization into the membrane of the nucleus, which
subsequently resulted in observed cell death (Porter
et al. 2007b).
An extremely special variant of the SEM is the
Environmental Scanning Electron Microscope
(ESEM), in which imaging can be performed with
a weaker vacuum (up to 20 torr) and thus allow a
gaseous environment in the specimen chamber. This
means the ability to examine wet, insulating samples,
thus allowing imaging to be conducted close to the
natural state of cells rather imaging under limitations
associated with vacuum conditions (Sayer et al.
1993). ESEM is thus destined to subsequently replace
the traditional SEM as such technological advances
have opened many new areas of application not pre-
viously accessible to SEM. This has been made pos-
sible due to on a number of changes to the instrument
of conventional SEM, namely the introduction of: (a)
Differential pumping, and (b) new detection systems
(Danilatos 1994; Uwins 1994; Griffith and Newbury
1996). ESEM has been previously used for the anal-
ysis of various cell lines and primary cells (Sonnichsen
and Donald 1997; McMenamin et al. 2003;
Misirli et al. 2007) but applications to using this
for nanoparticle-cell interactions have not been
investigated.
Electron-based imaging methods are extremely
powerful as the platforms are often combined with
spectroscopic-based techniques such as Electron
Energy Loss Spectroscopy (EELS) (Diociaiuti
2005) and Energy Dispersive X-ray analysis (EDX)
(Laszik et al. 1999; De Vizcaya-Ruiz et al. 2006) and
have the potential to yield elemental maps from thin
biological samples. The spectroscopic-based techni-
ques give complementary elemental/chemical infor-
mation and thus are capable of identifying ‘suspect’
nanoparticles. EDX is particularly sensitive to heavier
elements such as: P, S, K and Ca, whereas EELS are
sensitive to low atomic number elements such as C, N
and O as well as other elements with favourable
ionization cross-sections, such as Fe. EDX is easier
to use but EELS, although being the more difficult
technique, cannot only measure atomic composi-
tion but can also give information surrounding:
chemical bonding, valence and conduction band elec-
tronic properties, surface properties, and element-
specific pair distance distribution functions (Leapman
and Hunt 1991).
 8 R. Tantra & A. Knight
Summary and outlook
Summary and comparison of methods
The main focus in this section is to examine the
comparative advantages and disadvantages of the
various techniques, with the purpose of identi-
fying a technique that is most suitable to probe
cell-nanoparticle interactions. Table I presents a sum-
mary of the pros/cons of the techniques presented in
this paper; the techniques can be broadly categorized
into: (a) Electron microscopy, (b) fluorescence
microscopy, and (c) scanning probe microscopy. In
order to identify the technique most suitable for the
Nanotoxicology Downloaded from informahealthcare.com by Charing Cross & Westminster Medical Sch on 01/14/11
purpose of cell-nanoparticle interactions, each tech-
nique is matched against the specific criteria that have
been previously established.
It is clear from Table I that no perfect technique
exists, as there are clear limitations associated with
each. As is so often the case, the selection of the best
technique will be very much dependent on the kind of
information required and in turn will depend on the
types of questions that a particular research project
wishes to address.
Optical imaging techniques will mean the ability to
conduct live cell imaging, in which both surface and
sectioned images can be acquired; the latter main-
For personal use only.
tains imaging of live cells on the microscope stage
through the ability to perform optical sectioning. The
main limitation of such techniques however, is in the
choice of the type of nanoparticles that can be used
for nanotoxicological investigations. The option
to label the nanoparticles (e.g., using fluorescent
markers) is considered to be highly impractical
and as toxicological effects will be governed by
nanoparticle-label complex rather than the nanopar-
ticles themselves, such sample preparation should be
highly discouraged as it will defeat the purpose of the
study.
As with optical imaging, surface probe microscopy
based tools are also useful for live cell imaging as
analysis can be done under ambient conditions.
Unlike optical imaging, the library of nanoparticles
used for nanotoxicological investigations are not so
restricted. However, these tools are predominantly a
surface (or near surface) based technique and so, will
be suitable for analysis of whole cells rather than sub-
sectioned cells. The scanning tip can also be the
cause of potential artefacts in the image. In partic-
ular, surface forces between the tip and nanoparticles
can potentially remove those nanoparticles that are
weakly binding (Munz et al. 2008) on the surface of
cells.
When live cell imaging is not required, electron-
based imaging tool is the method of choice, as they
are suited for analysis of whole and sub-sectioned
cells. Such platforms are extremely powerful as
potentially they can offer complementary chemical
information, which is important to provide positive
identification of the nanoparticles under study. Con-
tamination (both chemical/biological in nature) can
arise from a variety of different sources, most
likely due through the use of unclean labware and
hands, during the handling/sample preparation of
nanoparticles. Recently, it was found that the endo-
toxin lipopolysaccharides (LPS) were identified as
‘contamination candidate’ (Vallhov et al. 2006) and
were shown to exist when nanoparticles are pro-
duced in a semi-clean environment. The presence
of LPS can affect results arising from bioassay type
tests, thus resulting in erroneous conclusions. The
presence of LPS can affect phenotype and function
of dendritic cells (DCs). DCs are highly present in
the lungs and the inhalation of contaminated parti-
cles may potentially lead to unwanted immune mod-
ulation (Vallhov et al. 2006). It is important
therefore to have tools that cannot only image but
also provide the chemical identification of contami-
nants such as LPS. The main limitation with
electron-based techniques is in their inability to
analyze under ambient and that extensive sample
preparation is required prior to analysis and will be
mainly governed by the skills of the operator, for
example protocol associated with the sectioning of
cells. The sectioning method can potentially intro-
duce image artefacts as sectioning can remove
loosely bound nanoparticles on the surface of the
cell and thus eventual migration into the centre of
the cell.
The new variants of tools in each of these catego-
ries have shown huge promise to overcome some of
the limitations found in the corresponding tradi-
tional technique. For example, super-resolution
techniques (Hell) have addressed the resolution
problem (Fernández-Suárez and Ting 2008; Irvine
et al. 2008). SNFUH (Scanning Near Field Ultra-
sound Holography), which has the great advantage of
being label-free, was shown to be able to probe
structural features below the surface of the cell.
New variants of the electron imaging techniques,
particularly ESEM is important as they minimize
the need to severely processing the sample prior to
imaging; this thus lowers the risk of generating arte-
facts (e.g., addition of metal coating) associated with
traditional SEM technique. Although such variant
techniques seems to hold immense potential
benefits, these tools are extremely novel and not
well established, particularly their use in relation
to nanotoxicological investigations have not been
investigated.
 Nanotoxicology Downloaded from informahealthcare.com by Charing Cross & Westminster Medical Sch on 01/14/11
For personal use only.

Table I. Summary of imaging techniques and their suitability to study nanoparticle-cell interactions, in accordance to the list of criteria as detailed in the text.

References
High in relation to
sensitivity Need Routine use, Whole + nanoparticle-cell
High [spatial Intracellular to label Non-invasive i.e., fast Live cell sectioned Chemical interaction type
Instrument selectivity resolution] detection nanoparticles? methods analysis time imaging cells? identification studies

Fluorescence Traditional Yes Sub-micron Yes Unless particles Yes, unless particles Salonen et al. 2008;
microscopy fluorescence are inherently requires (fluorescent) Yes Yes Yes No Gupta and Curtis 2004;
spectroscopy, fluorescent, labelling De la Fuente 2007;
e.g., confocal analysis requires Gorelik et al. 2002;
laser fluorescence (fluorescent) Dailey et al. 2003;
label Kemp et al. 2008;
Liu et al. 2004;
Calvo et al. 1994;
Lakowicz 1994

Multi-photon Sub-micron Glaschick et al. 2007

spectroscopy,
e.g., 2PEM

Super-resolution Tens of nm None

(fluorescence)

Scanning AFM Sub-nm Surface only No Yes, No, but does not Yes Whole cells Currently no, but Vasir and Labhasetwar
probe (~ 0.5 nm) but potential for require the same only scanning tip can 2008;
microscopy scanning tip extent of sample result in chemical Leroueil et al. 2007;
to interact with preparation needed readout, e.g., tip Leonenko et al. 2007
nanoparticles for electron enhanced Raman
SNFUH Sub-nm during scanning microscopy imaging spectroscopy – but Tetard et al. 2008;
based techniques the technique is Shekhawat and
not applicable Dravid 2005
SICM Sub-nm Below (near) surface to live cell imaging.
None

Electron SEM Few nm Yes, potentially the No No: Only for whole cells. No
microscopy (~ 2 nm) need for staining a) high vacuum Extensive sample Yes – through the Panessa-Warren
b) sectioning methods preparation required use of spectroscopic et al. 2008;
(potentially need for sectioning of cells methods such as Gupta and Curtis 2004;
for staining) and need to EDX and EELS de la Fuente et al. 2007
skilled operators. being on the same
platform
TEM Sub-nm No, due to extensive Verma et al. 2008;
(~ 0.2 nm) sample preparation Rothen-Rutishauser
and the need for et al. 2006;
skilled operators Nativo et al. 2008;
Kaul and Amiji 2005

ESEM Sub-nm
Yes
No: None
(a) not ambient but
under low vacuum
(b) sectioning methods
(potentially the
need for staining)

EFTEM Sub-nm Yes No: Thomas 2008;

(a) high vacuum Porter et al. 2006;
STEM Tens of nm (b) sectioning 2007a, 2007b
Review of imaging techniques
9
 10 R. Tantra & A. Knight
Looking to the future
It is evident that there is a need to assess nanoparticle
behaviour at a cellular level, if the underlying mechan-
isms of toxicity are to be understood in detail.
Undoubtedly, the use of imaging tools to probe
such mechanisms is vital. This paper has presented
a review of the various imaging tools that have been
used by past workers and the benefits/pitfalls of the
different techniques were assessed against a list of
criteria, developed for this purpose. We conclude
that tools under the ‘state-of-the-art’ umbrella
(EFTEM, STEM, ESEM, SNFUH, SICM and
‘super-resolution’ fluorescence microscopy) promise
Nanotoxicology Downloaded from informahealthcare.com by Charing Cross & Westminster Medical Sch on 01/14/11
to become the preferred method of choice, overcom-
ing some of the limitations posed by their traditional
counterparts. Electron-based imaging tools such as
EFTEM and STEM have resulted in the ability to
capture the image with enhanced contrast. In fluo-
rescence microscopy, the super-resolution techniques
have circumvented the limits imposed by diffraction.
However, such techniques are novel and far from
being used routinely. There is a real need to further
develop the use of such techniques for cellular
imaging.
Choosing the right technique and developing a pro-
tocol in order to give us the desired information about a
For personal use only.
given system is important to understand nanoparticle-
cell interaction. However, it is important that data
acquired is reliable. There is currently still a need to
standardize measurement protocols and the develop-
ment of suitable, well characterized reference mate-
rials for specific methods is very much required. For
some parameters such as particle size, internationally
accepted protocols and reference materials exist, which
in a limited number of cases have been standardized by
internationally standards organizations, such as Inter-
national Organization for Standardization (ISO),
European Committee for Standardisation (CEN),
Organisation for Economic Co-operation and Devel-
opment (OECD) and American Society for Testing
and Materials (ASTM). However, these standardized
protocols exist for well defined, simple systems and
often not applicable to real world toxicological testing
scenarios. Several issues hinder the establishment of
validated protocols that are very much needed for such
a purpose. These issues include the need to:
(a) Establish some kind of measurement guidelines
from the ‘end users’. In other words there is a
need to have an estimate on the required degree
of accuracy and reproducibility.
(b) Develop specific protocols to result in ‘fit for
purpose scenario’. It is important to establish a
validated protocol of the whole process rather
than separate entities, as accuracy/reproducibil-
ity will undoubtedly vary from assay to assay.
Currently, a collection of related ISO docu-
ments exists, which may help in the development
of specific protocols.
(c) Have reference standards, in which the ‘fit for
purpose’ considerations will be central to the
development of such reference materials.
Generally, the demand for reference materials
exceeds supply; this is both in terms of range of
materials and their corresponding availability. Cur-
rently, a database covering reference materials are
prepared by CITAC (Cooperation on International
Traceability in Analytical Chemistry) and ISO
REMCO (International Organization for Standardi-
zation [ISO 2000], Committee on Reference Materi-
als) (Rasberry and Monti 1996). The main role of
reference materials for nanoparticle measurements is
used to check the correct operations of the instru-
ments, which is particularly useful for troubleshooting
when unexpected results are obtained. However,
there is no doubt that a demand exists for reference
materials that can be used for specific toxicological
tests. A start in the right direction is the development
of reference materials in biologically-related media
and for such reference material to be: Accurate,
stable, easy to use and affordable. NIST (National
Institute of Standards and Technology) has taken the
first step towards achieving a critical path for reference
material development, with the introduction of gold
standards (RM 8011, RM 8012 and RM 8013, for 10,
30 and 60 nm in diameter, respectively) in citrate-
stabilized buffer. However, there is still a need to
validate the use of such reference material for specific
toxicological tests conducted, for example, if it is to be
used as potential reference material to qualitatively
test elemental analysis within subcellular compart-
ments. Issues such as the uncertainties associated
with changes in properties of the reference material
as a result of nanoparticle digestion into the cells and
possible loss of particles, must be addressed as part of
this validation process.
Acknowledgements
This work is financed under P5 (Bio-nanoparticle)
project, a DIUS (Department for Innovation,
Universities & Skills) NMS (National Measurement
System) chemical and biological metrology supported
programme. The authors would like to thank Drs
Anna Hills, Adrian Horgan, Neil Harrison and
Mr Baptiste Lamarre for helpful comments and
encouragement.

Declaration of interest: The authors report no
conflicts of interest. The authors alone are responsible
for the content and writing of the paper.
References
Calvo P, Thomas C, Alonso MJ, Vilajato JL, Robinson JR. 1994.
Study of the mechanism of interaction of poly(epsilon-
caprolactone) nanocapsules with the cornea by confocal laser-
scanning microscopy. Int J Pharmaceut 103(3):283–291.
Clarke DR. 1970. Review: Image contrast in the scanning electron
microscope. Chem Materials Sci 5(8):689–708.
Cognard, C, Constantin, B, Duclohier, H and Sebille, S. 2008.
Membrane surface topographs obtained by scanning ion con-
ductance microscopy (SICM): A new technique to investigate
Nanotoxicology Downloaded from informahealthcare.com by Charing Cross & Westminster Medical Sch on 01/14/11
pathophysiological phenotype development or dystrophin-
deficient muscle cells? Fundam Clin Pharmacol 22:240.
Conroy, J, Byrne SJ, Gun’ko YK, Rakovich YP, Donegan, JF,
Davies A, Kelleher D, Volkov Y. 2008. CdTe nanoparticles
display tropism to core histones and histone-rich cell organelles.
Small 4(11):2006–2015.
Dailey LA, Kleemann E, Wittmar M, Gessler T, Schmehl T,
Roberts C, Seeger W, Kissel T. 2003. Surfactant-free, biode-
gradable nanoparticles for aerosol therapy based on the branched
polyesters, DEAPA-PVAL-g-PLGA. Pharmaceut Res 20
(12):2011–2020.
Danilatos GD. 1994. Environmental scanning electron-microscopy
and microanalysis Mikrochimica Acta 114:143–155.
de la Fuente JM, Alcantara D, Penades S. 2007. Cell response to
magnetic glyconanoparticles: Does the carbohydrate matter?
IEEE Transact Nanobiosci 6(4):275–281.
For personal use only.
De Vizcaya-Ruiz A, Gutierrez-Castillo, ME, Uribe-Ramirez M,
Cebrian ME, Mugica-Alvarez V, Sepulveda J, Rosas I,
Salinas E, Garcia-Cuellar C, Martinez F, Alfaro-Moreno E,
Torres-Flores V, Osornio-Vargas A, Sioutas C, Fine PM,
Singh M, Geller MD, Kuhn T, Miguel AH, Eiguren-
Fernandez A, Schiestl RH, Reliene R, Froines J. 2006. Char-
acterization and in vitro biological effects of concentrated
particulate matter from Mexico City. Atmospheric Environ
40:S583–592.
Diociaiuti M. 2005. Electron energy loss spectroscopy microanal-
ysis and imaging in the transmission electron microscope: Exam-
ple of biological applications. J Electron Spectroscopy Related
Phenom 143(2–3):189–203.
Dufour P, Dufour S, Castonguay A, McCarthy N,
De Koninck Y. 2006. Two-photon laser scanning fluorescence
microscopy for functional cellular imaging: Advantages and
challenges or one photon is good. . . but two is better! M
S-Med Sci 22(10):837–844.
Dykstra MJ. 1993. A manual of applied techniques for biological
electron microscopy. Berlin: Springer-Verlag.
Edwards MA, Williams CG, Whitworth AL, Unwin PR. 2009.
Scanning ion conductance microscopy: A model for experimen-
tally realistic conditions and image interpretation. Analyt Chem
81(11):4482–4492.
Fernández-Suárez M, Ting AY. 2008. Fluorescent probes for
super-resolution imaging in living cells. Nature Rev Molec
Cell Biol 9(12):929–943.
Ferrara DE, Weiss D, Carnell PH, Vito RP, Vega D, Gao XH,
Nie, SM. 2006. Quantitative 3D fluorescence technique for the
analysis of en face preparations of arterial walls using quantum
dot nanocrystals and two-photon excitation laser scanning
microscopy. Am J Physiol-Regulat Integrat Comparat Physiol
290(1):R114–123.
Review of imaging techniques 11
Glaschick S, Rocker C, Deuschle K, Wiedenmann J, Oswald F,
Mailander V, Nienhaus GU. 2007. Axial resolution enhance-
ment by 4Pi confocal fluorescence microscopy with two-photon
excitation. J Biolog Phys 33(5–6):433–443.
Gorelik J, Ali NN, Kadir SHSA, Lab M, Stojkovic P, Armstrong L,
Sviderskaya EV, Negulyaev YA, Klenerman D, Bennett DC,
Lako M, Harding SE, Stojkovic M, Korchev YE. 2008. Non-
invasive imaging of stem cells by scanning ion conductance
microscopy: Future perspective. Tissue Engineer Part C –
Meth 14(4):311–318.
Gorelik J, Shevchuk A, Ramalho M, Elliott M, Lei C, Higgins CF,
Lab MJ, Klenerman D, Krauzewicz N, Korchev Y. 2002. Scan-
ning surface confocal microscopy for simultaneous topograph-
ical and fluorescence imaging: Application to single virus-like
particle entry into a cell. Proce Nat Acad Sci USA 99
(25):16018–16023.
Gorelik J, Zhang YJ, Shevchuk AI, Frolenkov GI, Sanchez D,
Lab MJ, Vodyanoy I, Edwards CRW, Klenerman D,
Korchev YE. 2004. The use of scanning ion conductance
microscopy to image A6 cells. Molec Cellular Endocrinol 217
(1–2):101–108.
Griffith E, Newbury DE. 1996. Introduction to environmental
scanning electron microscopy issue. Scanning 18(7):465–465.
Gupta AK, Curtis ASG. 2004. Lactoferrin and ceruloplasmin
derivatized superparamagnetic iron oxide nanoparticles for tar-
geting cell surface receptors Biomaterials 25(15):3029–3040.
Halbhuber KJ, Konig K. 2003. Modern laser scanning microscopy
in biology, biotechnology and medicine. Ann Anatomy-
Anatomischer Anzeiger 185(1):1–20.
Hayat MA. 2000. Principles and techniques of electron microscopy:
Biological applications. 4th ed. Cambridge, UK: Cambridge
University Press.
Hell S. 2008. Microscopy and its focal switch. Nature Meth
6(1):24–32.
International Organization for Standardization, Committee on Ref-
erence Materials (ISO REMCO). 2000. ISO 14887. Sample
preparation – dispersing procedures for powders in liquids.
Irvine SE, Staudt T, Rittweger E, Engelhardt J, Hell SW. 2008.
Direct light-driven modulation of luminescence from Mn-doped
ZnSe quantum dots. Angewandte Chem Int Ed 47(14):
2685–2688.
Kaul G, Amiji M. 2005. Cellular interactions and in vitro
DNA transfection studies with poly(ethylene glycol)-modified
gelatin nanoparticles. J Pharmaceut Sci 94(1):184–198.
Kemp SJ, Thorley AJ, Gorelik J, Seckl MJ, O’Hare MJ, Arcaro A,
Korchev Y, Goldstraw P, Tetley TD. 2008. Immortalization of
human alveolar epithelial cells to investigate nanoparticle uptake.
Am J Respirat Cell Molec Biol 39(5):591–597.
Korchev Y, Sanchez D, Korchev Y, Sanchez D, Anand P,Gorelik J,
Zhang YJ, Shevchuk A, Vodyanoy I, Lab M, Klenerman, D.
2007. ‘Non-contact’ mechanical characterization and stimula-
tion of cells using scanning ion conductance microscopy Biophys
J 33A–33A.
Korchev YE, Bashford CL, Milovanovic M, Vodyanoy I,
Lab MJ. 1997. Scanning ion conductance microscopy of living
cells. Biophys J 73(2):653–658.
Korchev YE, Raval M, Lab MJ, Gorelik J, Edwards CRW,
Rayment, T, Klenerman D. 2000. Hybrid scanning ion con-
ductance and scanning near-field optical microscopy for the
study of living cells. Biophys J 78(5):2675–2679.
Lakowicz JR. 1994. Topics in fluorescence spectroscopy: Probe
design and chemical sensing. Berlin: Springer.
Laszik A, Weisser HJ, Keresztury L, Pollak S, Papp G,
Pozsgai I. 1999. PCR typing of human semen stains after
SEM-EDX examination. Int J Legal Med 112(6):376–379.
 12 R. Tantra & A. Knight
Leapman RD, Hunt JA. 1991. Comparison of detection limits for
EELS and EDXS. Microscop Microanal Microstruct 2
(2–3):231–244.
Leonenko ZE, Finot E, Amrein M. 2007. Adhesive interaction
measured between AFM probe and lung epithelial type II cells.
Ultramicroscopy 107(10–11):948–953.
Leroueil PR, Hong SY, Mecke A, Baker JR, Orr BG,
Holl MMB. 2007. Nanoparticle interaction with biological
membranes: Does nanotechnology present a Janus face? Acc
Chem Res 40(5):335–342.
Li X. 2005. AFM imaging of water, cells and tissues. Materials
Research Society meeting proceedings. Symposium L – struc-
ture and mechanical behavior of biological materials. Warren-
dale PA, USA. Materials Research Society, L4.1.1–L4.1.6.
Limbach LK, Li YC, Grass RN, Brunner TJ, Hintermann MA,
Muller M, Gunther D, Stark WJ. 2005. Oxide nanoparticle
uptake in human lung fibroblasts: Effects of particle size,
Nanotoxicology Downloaded from informahealthcare.com by Charing Cross & Westminster Medical Sch on 01/14/11
agglomeration, and diffusion at low concentrations. Environ
Sci Technol 39(23):9370–9376.
Liu P, Zhang A, Zhou M, Xu Y, Xu LX. 2004. Real time 3D
detection of nanoparticle liposomes extravasation using laser
confocal microscopy. Engineering in Medicine and Biology
Society. IEMBS 2004. 26th annual international conference
of the IEEE.
Mann SA, Hoffmann G, Hengstenberg A, Schuhmann W,
Dietzel ID. 2002. Pulse-mode scanning ion conductance
microscopy – a method to investigate cultured hippocampal
cells. J Neurosci Meth 116(2):113–117.
Matsumoto B, Wilson L. 2002. Cell biological applications of
confocal microscopy Methods in Cell Biology. Amsterdam:
Elsevier Science & Technology Books.
McMenamin PG, Wealthall RJ, Deverall M, Cooper SJ,
For personal use only.
Griffin B. 2003. Macrophages and dendritic cells in the rat
meninges and choroid plexus: Three-dimensional localisation
by environmental scanning electron microscopy and confocal
microscopy. Cell Tiss Res 313(3):259–269.
Michalet X, Siegmund OHW, Vallerga JV, Jelinsky P, Millaud JE,
Weiss S. 2007. Detectors for single-molecule fluorescence imag-
ing and spectroscopy. J Modern Optics 54(2–3):239–281.
Misirli Z, Oner ET, Kirdar B. 2007. Real imaging and size values of
Saccharomyces cerevisiae cells with comparable contrast tuning to
two environmental scanning electron microscopy modes. Scan-
ning 29(1):11–19.
Munz M, Cox DC, Cumspson PJ. 2008. Nano-scale shear mode
testing of the adhesion of nanoparticles to a surface-support.
Physica Status Solidi A – Applic Mater Sci 205(6):1424–1428.
Nativo P, Prior IA, Brust M. 2008. Uptake and intracellular fate of
surface-modified gold nanoparticles. Acs Nano 2(8):1639–1644.
Pan Y, Brown A, Brydson R, Warley A, Li A, Powell J. 2006.
Electron beam damage studies of synthetic 6-line ferrihydrite
and ferritin molecule cores within a human liver biopsy. Micron
37(5):403–411.
Panessa-Warren, BJ, Warren JB, Maye MM., Van der Lelie D,
Gang O, Wong SS, Ghebrehiwet B, Tortora GT,
Misewich JA. 2008. Human epithelial cell processing of carbon
and gold nanoparticles. Int J Nanotechnol 5(1):55–91.
Porter AE, Gass M, Muller K, Skepper JN, Midgley P,
Welland M. 2007a. Visualizing the uptake of C60 to the cyto-
plasm and nucleus of human monocyte-derived macrophage
cells using energy-filtered transmission electron microscopy
and electron tomography. J Environ Sci Technol 15(8):
3012–3107.
Porter AE, Gass M, Muller K, Skepper JN, Midgley PA.,
Welland M. 2007b. Direct imaging of single-walled carbon
nanotubes in cells. Nature Nanotechnol 2(11):713–717.
Porter AE, Muller K, Skepper J, Midgley P, Welland M. 2006.
Uptake of C60 by human monocyte macrophages, its localiza-
tion and implications for toxicity: Studied by high resolution
electron microscopy and electron tomography. Acta Biomater
2(4):409–419.
Rasberry SD, Monti CL. 1996. Worldwide production of CRMs:
1996 Status Report, NIST Report; Feb 1996.
Rodenburg JM. 1997. Electron microscopy and analysis 1997:
Proceedings of the Institute of Physics Electron Microscopy
and Analysis Group Conference. Institute of Physics Conference
Series, Cavendish Laboratory, University of Cambridge.
Rothen-Rutishauser BM, Schurch S, Haenni B, Kapp N,
Gehr P. 2006. Interaction of fine particles and nanoparticles
with red blood cells visualized with advanced microscopic tech-
niques. Environ Sci Technol 40(14):4353–4359.
Salonen E, Lin S, Reid ML, Allegood M, Wang X, Rao AM,
Vattulainen I, Ke PC. 2008. Real-time translocation of fullerene
reveals cell contraction. Small 4(11):1986–1992.
Sayer M, Nolan P, Hansson CM. 1993 Scanning electron-
microscopy without pain – the environmental SEM. Canad
Ceramics Quart – J Canadian Ceramic Soc 62(2):104–105.
Schwarz H, Humbel BM. 2008. Preparation of biological samples
for electron microscopy. Proceedings of the 14th European
Microscopy Congress; Aachen, Germany, 1–5 September
2008. Berlin: Springer.
Shekhawat GS, Dravid VP. 2005. Nanoscale imaging of buried
structures via scanning near-field ultrasound holography. Sci-
ence 310(5745):89–92.
Shevchuik AI, Frolenkov GI, Sanchez D, James PS, Freedman N,
Lab MJ, Jones R, Klenerman D, Korchev YE. 2006. Imaging
proteins in membranes of living cells by high-resolution scanning
ion conductance microscopy. Angewandte Chemie – Int Ed
45(14):2212–2216.
Sonnichsen C, Donald A. 1997. Studies on living cells using
environmental scanning electron microscopy. Eur J Cell Biol
74:102–102.
Su TJ, Theofanidou E, Arlt J, Dryden DTF, Crain J. 2004. Single
molecule fluorescence imaging and its application to the study of
DNA condensation. J Fluoresc 14(1):65–69.
Tetard L, Passian A, Venmar KT, Lynch RM, Voy BH,
Shekhawat G, Dravid VP, Thundat T. 2008. Imaging nanopar-
ticles in cells by nanomechanical holography. Nature Nanotech-
nol 3(8):501–505.
Uwins PJR. 1994. Environmental scanning electron-microscopy.
Materials Forum 18:51–75.
Vallhov H, Qin J, Johansson SM, Ahlborg N, Muhammed MA,
Scheynius A, Gabrielsson S. 2006. The importance of an endo-
toxin-free environment during the production of nanoparticles
used in medical applications. Nano Lett 6(8):1682–1686.
Vasir JK, Labhasetwar V. 2008. Quantification of the force
of nanoparticle-cell membrane interactions and its influence
on intracellular trafficking of nanoparticles. Biomaterials
29(31):4244–4252.
Verma A, Uzun O, Hu YH, Hu Y, Han HS, Watson N, Chen SL,
Irvine DJ, Stellacci F. 2008. Surface-structure-regulated cell-
membrane penetration by monolayer-protected nanoparticles.
Nature Mater 7(7):588–595.
Warheit DB, Reed KL, Sayes CM. 2009. A role for nanoparticle
surface reactivity in facilitating pulmonary toxicity and develop-
ment of a base set of hazard assays as a component of nanopar-
ticle risk management. Inhal Toxicol 21(S1):61–67.
Wiesendanger R. 1998. Scanning probe microscopy: Analytical
methods (NanoScience and technology). Berlin: Springer.
Williams DB, Carter CB. 2009 Transmission electron microscopy:
A textbook for materials science. 2nd ed. Berlin: Springer.