LETTERS
Direct imaging of single-walled carbon
nanotubes in cells
ALEXANDRA E. PORTER1*, MHAIRI GASS2, KARIN MULLER3, JEREMY N. SKEPPER3,
PAUL A. MIDGLEY4 AND MARK WELLAND1
1
The Nanoscience Centre, University of Cambridge, 11 J. J. Thompson Avenue, Cambridge CB3 OFF, UK
UK SuperSTEM, Daresbury Laboratory, Daresbury, Cheshire WA4 4AD, UK
3
Multiimaging Centre, Department of Anatomy, University of Cambridge, Downing Street, Cambridge CB2 3DY, UK
4
Department of Materials Science and Metallurgy, University of Cambridge , Pembroke Street, Cambridge CB2 3QZ, UK
*e-mail: [email protected]
2
Published online: 28 October 2007; doi:10.1038/nnano.2007.347
The development of single-walled carbon nanotubes for various
biomedical applications is an area of great promise. However, the
contradictory data on the toxic effects of single-walled carbon
nanotubes1–10 highlight the need for alternative ways to study
their uptake and cytotoxic effects in cells. Single-walled carbon
nanotubes have been shown to be acutely toxic1–3 in a number
of types of cells, but the direct observation of cellular uptake
of single-walled carbon nanotubes has not been demonstrated
previously due to difficulties in discriminating carbon-based
nanotubes from carbon-rich cell structures. Here we use
transmission electron microscopy and confocal microscopy to
image the translocation of single-walled carbon nanotubes
into cells in both stained and unstained human cells. The
nanotubes were seen to enter the cytoplasm and localize
within the cell nucleus, causing cell mortality in a dosedependent manner.
Single-walled carbon nanotubes (SWNTs) are hydrophobic,
tubular nanostructures with diameters of only 0.6–3.5 nm, and are
therefore much smaller than the nuclear pore complex but within
the size range of other pores such as ion channels and gap
junction complexes that allow molecules to move freely into and
out of cells. It has therefore become of great concern that SWNTs
may also enter cells through the lipid bilayer and interact with
organelles or even enter the nucleus11,12. SWNTs are routinely
synthesized using a metal catalyst, which also has the potential to
have toxic effects on cells1,8,13. Internalization of fluorochromelabelled SWNTs into cells has been observed with confocal
microscopy9. However, tracing the same process using unlabelled
SWNTs is challenging, because it is difficult to distinguish carbonbased nanotubes from carbon-rich organelles due to similarities in
composition and dimensions1. Visualizing SWNTs inside cells will
help us understand how SWNTs enter cells, where they migrate to,
and their fate after uptake. Previously we have demonstrated that
low-loss energy-filtered transmission electron microscopy
(EFTEM) enables clear differentiation between C60 and cellular
compartments in unstained sections14; however, there has been no
information about the pathological effect of internalized C60 on
cell morphology and survival. Here we visualized individual
SWNTs in the cell using EFTEM in combination with electron
energy loss (EEL) spectrum imaging—a method that yields
characteristic energy-loss information—and mapped the
distribution of SWNTs in the cell. This technique gives a good
image contrast of unstained sections that cannot be achieved using
conventional imaging techniques15–17. We also present a simple
method for imaging intracellular SWNTs using the confocal
microscope by filling the nanotubes with silver iodide (AgI) as
described in ref. 18. Encapsulating AgI inside SWNTs increases the
reflectance from SWNTs without chemically modifying the surface
and also removes toxicity issues19,20. Structural changes seen in the
cell were correlated to the distribution of internalized SWNTs, and
these changes were compared with the results obtained from
conventional cell viability assays.
Macrophages, which are cells of the immune system that can
cause inflammation, form the first line of defence against foreign
materials in many tissues, including the lung. A significant
proportion of nanoparticles entering the pulmonary airways are
likely to be ingested by macrophages, where they are
concentrated into membrane-bound organelles, phagosomes or
lysosomes, before entering the blood and lymph circulation.
Therefore, we chose human monocyte-derived macrophages
(HMMs) as an in vitro model for the exposure of SWNTs to
cells. The HMMs were treated with SWNTs for 2 and 4 days at
concentrations of 0–10 mg ml21. TEM studies were performed at
a concentration of 5 mg ml21. Following incubation with SWNTs,
cell viability was measured in parallel using neutral red (NR)
(3-amino-7-dimethylamino-2-methyl-phenazine hydrochloride)
and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide] assays. The HMMs exposed to concentrations up to
10 mg ml21 after 2 days showed no significant toxicity with the
NR assay, but significantly fewer cells survived after 4 days in
both assays. The NR assay showed a significant decrease in cell
viability at the highest concentrations of SWNTs. In the MTT
assay a significant decrease in cell viability was found at all
concentrations above 0.3 mg ml21. The localized effect of the
uptake of SWNTs on cell death was quantified by indexing 100
cells in each group as either apoptotic, necrotic or healthy. No
significant difference in cell death was observed between the
control and SWNT-treated cells at 2 days. This difference became
significant after 4 days (Fig. 1b).
Traditional TEM studies use heavy metal stains to generate
contrast and reveal the structure of membranes and subcellular
organelles. This obscures visualization of the intracellular
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LETTERS
120
% cell viability
100
HAADF
a
NR, 4 days
a
ab
NR, 2 days
ab
a
b
bc
a
cd
80
BF
MTT, 4 days
ab
cd
b
d
Spectrum image
d
200 nm
60
Fe
40
Ep
5 nm
20
0
0
0.3125
0.625
1.25
2.5
5
10
0.5%
THF
m
c
SWNT (μg ml–1)
30
Necrotic
25
SWNT
% dead cells
20
1,000 nm
A
SWNT
100 nm
3
5
2
NA
SWNT
2 days
6
2
1
NA
nm
SWNT
SWNT
4 days
200 nm
Figure 1 Toxicity of SWNTs. a, SWNT-treated cells show decreased viability in
the neutral red (NR) assay at 4 days when compared to control cells (NA)
(ANOVA, F ¼ 4.406 and p , 0.001). Fishers PLSD indicated significantly
decreased viability at 2.5 and 10 mg ml21 when compared with equivalent
controls (groups labelled with the same letters indicate homogeneous means).
With the MTT assay, at all concentrations, SWNT-treated cells show significantly
decreased viability when compared to control cells (NA) at 4 days (ANOVA,
F ¼ 16.67, p , 0.0001). b, TEM index of the analysis of cell death (apoptosis/
necrosis; +statistical error %) between control (NA) and SWNT-treated cells
(5 mg ml21). Numbers indicate % dead cells.
SWNTs. Typically, we study the morphology of the cells in stained
sections, which are compared with unstained sections. Achieving
contrast from unstained sections is technically demanding,
but using low-loss EEL spectrum imaging (Fig. 2a) and low-loss
(0– 78 eV) EFTEM with a small energy window (2 –6 eV)
(Fig. 2c– f ), we achieved improved contrast between SWNTs and
certain cell organelles (plasma membrane, vesicles and the
nucleus) without staining (Fig. 2a –f ). High-resolution brightfield imaging (Fig. 2b) and low-loss EELS (see Supplementary
Information, Figs S1, S2) confirmed that these structures were
SWNTs. In EFTEM we observed SWNTs forming bundles with
dark contrast at 0 eV and reverse bright contrast at all energy
losses up to 78 eV (see Supplementary Information, Fig. S3). We
expect that contrast between the SWNTs and the cell arises
primarily from the higher carbon atom density of the SWNTs. In
EFTEM, this contrast was enhanced at 26 eV, corresponding to
the higher s þ p bulk plasmon from the graphitic nanotubes
with respect to the amorphous cell (see Supplementary
Information, Fig. S2)15–17.
2
SWNT
10
10
0
1,000 nm
18
15
5
l
n
Apoptotic
nm
2,000 nm
Figure 2 Intracellular distribution of SWNTs in unstained sections.
a, HAADF-STEM image of SWNTs within a lysosome (2 days exposure).
Box shows low-loss EELS image: Fe map (white); plasmon map (Ep) ranges from
17.5 eV (blue) to 21.5 eV (white). b, High-resolution bright-field (BF) image of
SWNTs. Arrows indicate the diameter of SWNTs. c – f, Low-loss EFTEM images
of cells (4 days exposure) at 26 eV, showing SWNTs fused with the plasma
membrane (c); at 75 eV, showing SWNTs parallel with the plasma membrane
and merging with lysosomes (d); at 26 eV, showing SWNTs crossing the nuclear
membrane (e); at 0 eV, showing SWNTs inside the nucleus (f). Inset to f shows
individual SWNTs. Labels: m, plasma membrane; l, lysosomes; nm, nuclear
membrane; c, cytoplasm; n, nucleus.
At 2 days, SWNTs were found within lysosomes (Fig. 2a,b). To
map the SWNTs, the position of the bulk plasmon peak was
extracted (see Supplementary Information, Fig. S1) and clearly
shows features not visible in the associated image (Fig. 2a). Maps
of the Fe M2,3 edge showed a significant number of iron clusters
associated with the SWNTs (An EELS M2,3 edge corresponds to
an energy loss of the primary beam because electrons from the
3p1/2 and 3p3/2 shells have been excited to empty states above the
Fermi level.).
At 4 days exposure, SWNTs had fused and aligned with their
long axes parallel to the plasma membrane (Fig. 2c). Bundles of
SWNTs were also found within long tubular vesicles, which are
probably early endosomes (Fig. 2d). In some regions these
oriented bundles merged with lysosomes and fused with or
crossed the lysosomal membrane (Fig. 2d; see also
Supplementary Information, Fig. S1). Most significantly, SWNTs
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LETTERS
nu
pm
m
SWNT
1 μm
500 nm
200 nm
100 nm
SWNT
Membrane
SWNT
1 μm
1 μm
5 nm
Membrane
SWNT
SWNT
SWNT
Healthy
membrane
200 nm
Disrupted
membrane
50 nm
50 nm
Figure 3 Cell morphology after exposure to SWNTs. a, A healthy cell exposed
to 5 mg ml21 SWNTs for 2 days. b, Zero-loss EFTEM image of a necrotic cell at
4 days. Upper right: higher magnification image of SWNT ( 3 – 6 nm diameter)
bundles around the plasma membrane (pm) shown in the boxed region of the
main panel. c, HAADF-STEM images of aggregates of SWNTs within a necrotic
cell, with opposite contrast to the zero-loss image in b. d, Higher magnification
of boxed area in image c.
translocated across the nuclear membrane (Fig. 2e) and localized
within the nucleus (Fig. 2f ).
Selected sections were stained with uranyl acetate and lead
citrate to enhance contrast from cell organelles and membranes.
In stained sections we noted a time-dependent change in
morphology. At 2 days (Fig. 3a) exposure to SWNTs, the
majority of cells appeared healthy, and high-angle annular darkfield scanning TEM (HAADF-STEM) showed little alteration in
morphology. In comparison, after 4 days exposure we observed
features characteristic of cell death (Fig. 3b –d). Cell death
occurred by two processes: apoptosis and necrosis. Apoptotic
cells appeared shrunken, with heavily capped chromatin.
Cytoplasmic and nuclear contents had leached out in necrotic
cells (Fig. 3b –d). A marked increase in cell mortality could be
correlated with regions of higher SWNT density.
The mechanism by which SWNTs enter the cell is not fully
understood and reports have indicated that they could either
traverse the cellular membrane by means of endocytosis12 or they
insert into and diffuse through the lipid bilayer11. In our
experiments, after 2 and 4 days exposure, the majority of SWNTs
were located within phagosomes and lysosomes of healthy cells
(Fig. 4a,b), suggesting uptake by phagocytosis—a process of
Figure 4 Localization of intracellular SWNTs within stained cell sections.
a, HAADF-STEM image showing SWNT bundles being actively ingested by a
phagosome at 4 days. b, Higher magnification image of boxed area in image
a illustrating that SWNTs were compartmentalized inside the phagosomal membrane
(m). c, Bright-field STEM image of SWNTs translocating across the lipid bilayer into
the neighbouring cytoplasm. d, Confocal microscope image of HMM exposed to
AgI@SWNT at 3 days, confirming inclusion of SWNT bundles inside the nucleus
(blue). e, HAADF-STEM image showing SWNTs within a lysosome, with membrane
disruption at the region where SWNTs fused with the membrane.
engulfment by which macrophages ingest cellular fragments or
micro-particles. SWNTs also translocated across the membrane
into the neighbouring cytoplasm at 4 days (Fig. 4c), indicating
passive uptake through the lipid bilayer. Furthermore, AgI-filled
SWNTs (AgI@SWNTs) were seen in the nucleus after 3 days
when imaged in reflectance mode on the confocal microscope
(Fig. 4d). In most regions the lysosomal membrane remained
continuous, but in some areas where SWNTs had fused with the
membrane, disruption was evident (Fig. 4e). This is the first
demonstration that individual SWNTs can cross lipid bilayers
and enter the cytoplasm and nucleus of the cell.
HAADF-STEM electron tomography of intracellular SWNTs
successfully confirmed, in a spatially resolved three-dimensional
image, that the SWNTs were located within cell organelles.
Figure 5 shows a representative tomogram illustrating SWNT
bundles within lysosomes.
In summary, we show that it is possible to map the location of
intracellular SWNTs using TEM and confocal microscopy. We
successfully imaged individual SWNTs within lysosomes and also
crossing cell membranes. We demonstrate two possible pathways
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LETTERS
Nanotechnologies. These SWNTs were produced by chemical vapour deposition
(CVD) by means of the HiPco process21. This process produces SWNTs with a
gaussian distribution of diameters, with a maximum peak around 0.9 – 1.2 nm.
SWNTs were fully characterized by high-resolution (HR)-TEM and EELS prior
to exposure, which showed bundles of nanotubes decorated with iron catalyst
particles and also onion-like structures around iron particles, and confirmed
they were graphitic-like carbon.
l
mi
500 nm
mi
mi
mi
CELL CULTURE STUDY
Figure 5 HAADF-STEM tomogram of bundles of SWNTs within lysosomes at
4 days in a stained cell section. a – d, A series of horizontal slices through a
HAADF-STEM reconstruction. The slices are 10 nm apart in the z-direction and
illustrate SWNT bundles (red arrows) within lysosomes (l) in cells containing
mitochondria that appear healthy. Variations in the mitochondrial matrix (mi) and
SWNT bundles were observed at different heights within the section. Subsequent
segmentation of SWNT bundles confirmed that they were continuous throughout
the section (see Supplementary Information, Movie).
The detailed methods used for the in vitro cell culture study and electron
microscopy have been published previously22. In brief, human monocytederived macrophages were treated with SWNTs for 2 and 4 days at
concentrations of 0 – 10 mg ml21. SWNTs were dispersed in tetrahydrofuran
(THF), and bundles were broken up by ultrasonication for 10 min. The THF was
used as a vehicle to hinder bacteria formation after 4 days in cell culture. SWNTs
were freshly suspended in the THF by sonicating for 30 min. The suspension was
immediately mixed into a serum free cell culture medium using a pipette. No
dispersing reagent (such as a surfactant) was used to break up the as-prepared
nanotube bundles, so as to maintain the intrinsic properties of the nanotubes.
Following the incubation with SWNTs, cell viability was measured using two cell
viability assay: (1) The NR assay (3-amino-7-dimethylamino-2-methylphenazine hydrochloride), which measures the lysosomal accumulation of NR
dye in viable cells, and (2) the MTT assay, which measures the intracellular
enzymatic conversion of MTT [3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide] by mitochondrial dehydrogenase into
formazan in viable cells. In both assays, cell viability is given as a relative measure
of toxicity, where the control sample is set to represent 100% cell viability. Each
experiment was performed in triplicate. Results were calculated as mean+
standard deviation. Statistical analysis was performed using ANOVA to identify
differences between groups followed by a post hoc Fishers protected least
significance difference test (PLSD) with significance set at p , 0.01. Groups
labelled with the same letters in Fig. 1a have homogeneous means.
ELECTRON MICROSCOPY
of entry of SWNTs into cells: energy-dependent phagocytosis or
endocytosis and passive diffusion across lipid bilayers. SWNTs
were found primarily within lysosomes, but more significantly
within the cytoplasm and the nucleus. Uptake to these sites
implies they may interact with intracellular proteins, organelles
and DNA, which would greatly enhance their toxic potential.
SWNTs also fused with the plasma membrane, where they have
been shown to cause cell damage through lipid peroxidation and
oxidative stress2,3. Localization of SWNTs at these sites was
correlated to an increase in cell death in both cell viability assays
and TEM analysis. However, TEM provided direct estimates of
cell viability and the simultaneous presence of SWNTs in the
same cell, while cell viability assays measure the change in
survival from a larger cell population (n ¼ 100 cells in TEM). NR
uptake or MTT formazan formation are both compared to a
control value set to 100%. These are both global assays of a large
number of cells (n † 0.05 106). The NR assay detects uptake of
the dye by lysosomes and does not discriminate between the
routes of cell death, merely giving an assay of viability. The MTT
assay gives false positives, as the SWNTs bind to the insoluble
formazan5, preventing its dissolution for subsequent
spectrophotometric assay. In contrast, TEM determines the mode
of cell death by clearly defined ultrastructural characteristics. To
conclude, we show that direct imaging of SWNTs within cells is
achievable and is essential to complement cytotoxicity assays to
understand localized effects of SWNTs and establishment of their
potential toxicity.
METHODS
MATERIALS
Owing to their widespread use and easy commercial availability, purified SWNTs
(purified HiPco, ,15 wt% ash content) were purchased from Carbon
4
TEM studies were performed at a concentration of 5 mg ml21 in accordance with
previous work14,22. For electron microscopy studies, SWNTs were fixed in 4%
gluteraldehyde in piperazine-1,4-bis(2-ethanesulphonic acid) (PIPES) buffer,
dehydrated, embedded, and subsequently sectioned with an ultramicrotome at
20 – 40 nm for high-resolution STEM imaging, 70 nm for EFTEM studies and
300 nm for three-dimensional electron tomography. The TEM sections were cut
onto lacy carbon film grids and bare 600-mesh copper TEM grids. Selected
sections were bulk stained with osmium tetroxide and post-stained with uranyl
acetate and lead citrate for 5 min in each to enhance contrast from cell
membranes and organelles. Changes in cell morphology upon exposure to
SWNTs were assessed by comparing the structure and morphology of exposed
and unexposed cells. Quantitative analysis of cell death was performed on
osmicated cells by indexing 100 cells in each group (2, 4 days exposed and
controls) as either apoptotic, necrotic or healthy. The statistical error was
estimated using a binomial approximation in which the error (e) for a confidence
level p ¼ 0.05 was estimated as e ¼ 1.96 SQRT[(P 1– P)]/n, where n ¼ 100
and P is the percent of cases observed. Apototic and necrotic cells were identified
by comparing cell morphology to reference images from previous studies23. To
categorize the cells, apoptotic cells were characterized as shrunken, containing
vacuoles in a condensed cytoplasm, and showing heavily capped chromatin.
Necrotic cells were more electron-lucent than either normal or apoptotic cells,
and their cytoplasmic and nuclear contents appeared to be leached out.
All TEM observations were made after viewing several hundred cell profiles
from three different exposures. EFTEM studies were performed on a Philips
CM300 operated at 300 kV with a Gatan imaging filter (GIF) model 2002 using a
10 mm objective aperture to optimize spatial resolution24. To optimize contrast,
zero-loss images of non-stained sections were taken using a 3 eV slit. Selecting an
energy window on the higher side of the plasmon feature enhanced the contrast
from the SWNTs. Zero-loss images of stained sections were taken using a 20 eV
slit. Low-loss energy filtered series were recorded from 0 to 30 eV using a 2 eV slit
and 2 eV step size14. The position of the bulk s þ p plasmon peak was extracted
from the EFTEM series using interactive data language (IDL) image
processing software.
High-resolution imaging and EELS were performed at the UK SuperSTEM
laboratory on a 100 kV VG HB501 dedicated STEM fitted with a Nion secondgeneration spherical aberration corrector and a Gatan Enfina EELS. The
convergence semi-angle of the electron probe was 24 mrad for both imaging and
spectroscopy. The collection semi-angle for the EELS was 19 mrad and
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LETTERS
Received 25 July 2007; accepted 27 September 2007; published 28 October 2007.
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Financial support was provided by the IRC in Nanotechnology (Cambridge, UK), the Isaac Newton Trust,
EPSRC, FEI Company, the Royal Academy of Engineering, the Leverhulme Trust for a Senior Research
Fellowship and the Oppenheimer Research Fellowship. The Multiimaging Centre was established with
funding from the Welcome Trust. SuperSTEM funding came from EPSRC grant no. EP/D040396/1. We
thank J. Bendell, S. Friedichs and Haibo E. for their informative discussions.
Correspondence and requests for materials should be addressed to A.E.P.
Supplementary information accompanies this paper on www.nature.com/naturenanotechnology.
70 – 210 mrad for HAADF imaging. Low-loss EELS spectrum images were
acquired at every pixel over a user-defined area with an energy dispersion of
0.1 eV per channel, covering an energy range of 0 – 100 eV. By fitting a
nonlinear least-squares (NLLS) fit to the plasmon peak, its peak position was
extracted and mapped. The iron M2,3 edge occurs at 54 eV, and background
fitting can be problematic due to effects from plural scattering and the plasmon
tail. By removing plural scattering and extracting the imaginary part of the
dielectric function, 12, from a Kramers – Kronig analysis, an improved
background fit was achieved. This in turn allows for a more accurate signal
integral of the iron M2,3 edge. Medium-resolution HAADF-STEM of stained
sections was performed on an FEI Tecnai F20 operated at 200 kV using a 30 mm
condenser aperture and a camera length of 150 mm.
In electron tomography, projections of the object are acquired from several
different orientations and reconstructed into a three-dimensional volume.
HAADF-STEM tomographic data sets were acquired over a tilt range of 2708 to
þ708 using a step size of 28. Three-dimensional reconstruction was carried out
using the simultaneous iterative reconstruction technique (SIRT)25,26 using
Inspect 3D image processing software. Reconstructions were visualized by a
voltex projection using Amira 3D visualization software (Mercury Computer
Systems, France).
CONFOCAL MICROSCOPY
For confocal microscopy, HMMs were exposed to AgI@SWNT at a concentration
of 5 mg ml21 for 3 days and imaged using a Leica TCS SP2 confocal microscope,
in reflectance mode. Three-dimensional image stacks were taken though the
z-direction of the cell to confirm inclusion of AgI@SWNTs within the cell
volume. Cells were mounted in Vectashield containing 40 ,6-diamidino2-phenylindole (DAPI, Vectalabs), a fluorescent stain that binds strongly
to DNA.
Author contributions
All authors conceived and designed the experiments: A.E.P. and P.A.M. performed the TEM and HAADFSTEM experiments and analysis, M.G. performed the aberration corrected STEM and EELS experiments,
K.M. performed the cell studies and confocal microscopy, A.E.P. and J.S. performed the EM preparation,
A.E.P., K.M. and M.G. analysed the data, and A.E.P. wrote the paper: All authors discussed the results.
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