Advanced Review

Cryo-electron microscopy and

cryo-electron tomography
of nanoparticles
Phoebe L. Stewart*

Cryo-transmission electron microscopy (cryo-TEM or cryo-EM) and cryo-

electron tomography (cryo-ET) offer robust and powerful ways to visualize nano-
particles. These techniques involve imaging of the sample in a frozen-hydrated
state, allowing visualization of nanoparticles essentially as they exist in solution.
Cryo-TEM grid preparation can be performed with the sample in aqueous sol-
vents or in various organic and ionic solvents. Two-dimensional (2D) cryo-TEM
provides a direct way to visualize the polydispersity within a nanoparticle prepa-
ration. Fourier transforms of cryo-TEM images can confirm the structural perio-
dicity within a sample. While measurement of specimen parameters can be
performed with 2D TEM images, determination of a three-dimensional (3D)
structure often facilitates more spatially accurate quantization. 3D structures can
be determined in one of two ways. If the nanoparticle has a homogeneous struc-
ture, then 2D projection images of different particles can be averaged using a
computational process referred to as single particle reconstruction. Alternatively,
if the nanoparticle has a heterogeneous structure, then a structure can be gener-
ated by cryo-ET. This involves collecting a tilt-series of 2D projection images for
a defined region of the grid, which can be used to generate a 3D tomogram.
Occasionally it is advantageous to calculate both a single particle reconstruction,
to reveal the regular portions of a nanoparticle structure, and a cryo-electron
tomogram, to reveal the irregular features. A sampling of 2D cryo-TEM images
and 3D structures are presented for protein based, DNA based, lipid based, and
polymer based nanoparticles. © 2016 Wiley Periodicals, Inc.
How to cite this article:
WIREs Nanomed Nanobiotechnol 2016. doi: 10.1002/wnan.1417

INTRODUCTION the sample that is imaged to correspond to a dis-

torted version of the original specimen.5 Cryo-TEM

C ryo-transmission electron microscopy (cryo-

TEM), also known as cryo-EM, was developed
in the 1980s as a way to visualize biological samples
sample preparation typically involves placing a drop-
let of the sample on an EM grid, blotting with filter
paper to remove excess water or solvent, and flash
in a near native state at the resolution of the trans- freezing the grid in a cryogen so that the solvent
mission electron microscope (0.1–2 nm).1–4 More forms a vitreous or amorphous, glass-like state.6–8
traditional transmission electron microscopy (TEM) The most commonly used type of grid has a carbon
preparation techniques involve drying and staining support film with holes in it, so that the sample parti-
or plastic embedding of biological samples, causing cles can be suspended in a layer of frozen solvent
within a hole of the support layer.6 Then cryo-TEM
images may be collected of the frozen sample without
*Correspondence to: pls47@case.edu any adsorption artifacts or background signal from
Case Western Reserve University, Cleveland, OH, USA the support layer. This plunge freezing preparation
Conflict of interest: The author has declared no conflicts of interest method preserves the specimen as a solid that can be
for this article. inserted into the ultra-high vacuum of a transmission

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 Advanced Review wires.wiley.com/nanomed

electron microscope. Achieving the vitreous state of After a tomogram has been generated it is sometimes
frozen water is important, as hexagonal or cubic ice, possible to computationally average repeating units
which would be formed by freezing water more within the overall structure with the technique of
slowly or at higher temperatures, expands during the sub-tomogram averaging.15 Data processing is an
freezing process. This expansion during freezing can important component of both single particle recon-
easily crush a biological or fragile specimen and dis- struction and cryo-ET. Numerous software packages
tort its structure. In addition, ice crystals can cause have been developed for generating 3D structures
image artifacts and make it difficult to interpret cryo- and analyzing them.16–37 Determining a 3D structure
TEM images.9 In cryo-TEM, the frozen sample grid not only provides a more complete representation of
is kept at liquid nitrogen temperature during imaging the sample than can be provided by a 2D image, but
in a transmission electron microscope. The beauty of it also facilitates more spatially accurate quantitative
cryo-TEM is that images can be generated from the measurements.
actual specimen in a frozen-hydrated state, which is Although beyond the scope of this review to
essentially as it exists in solution. cover in detail, it is important to note that cryo sam-
While a 2D cryo-TEM image of a biological ple preparation methods have also been developed
specimen or nanoparticle provides useful informa- for scanning electron microscopy (cryo-SEM).38,39
tion, the real power of cryo-TEM is that 3D struc- Applications range from animal and plant tissue, to
tures can be determined.8,10,11 In fact, over the past cosmetics and pharmaceuticals, as well as food indus-
several years there has been an enormous ‘resolu- try products. However in the case of SEM, the image
tion revolution’ that has resulted in dozens of struc- acquired is not a projection image, as is obtained in
tures at atomic resolution,12 up to 2.2 Å for TEM. By definition, TEM involves collection of elec-
β-galactosidase.13 Structures can be generated one of trons that have been transmitted through the sample.
two ways. If the specimen is structurally and chemi- In contrast, SEM images are generated from second-
cally homogeneous, like an icosahedral virus, an ary electrons and backscattered electrons created
approach called single particle reconstruction may be when a focussed electron beam is scanned across a
employed. This involves averaging thousands of specimen. When combined with focussed ion beam
cryo-TEM images of different particles in various (FIB) milling for serial block face imaging, cryo FIB-
orientations, usually random orientations, or as the SEM is a technique that enables generation of 3D
particles were oriented when the grid was frozen. volume representations.40
Alternatively, if the specimen is one-of-a-kind, like an Cryo-TEM methods have recently begun to be
irregularly shaped liposome, then cryo-ET may be applied to engineered nanoparticles.9,41–43 This
employed. Cryo-ET, and electron tomography in review provides examples of 2D cryo-TEM images
general, involve tilting the specimen through a large and 3D cryo-TEM structures of various types of
angular range, e.g., −70 to +70 , and collecting a tilt nanoparticles. The goal is to illustrate what is possi-
series of images of a single specimen area. For every ble with cryo-TEM imaging and to show the poten-
cryo-TEM sample the electron dose is a critical factor tial of cryo-TEM single particle reconstruction
that must be controlled. The sample region of interest and cryo-ET for imaging nanoparticles in three
is only exposed to a low electron dose (typically ~20 dimensions.
electrons/Å2) and only during image acquisition. In
tomography, the maximum dose has to be divided by
the total number of images in the tilt series. This
CHALLENGES IN CRYO-TEM
makes the individual images rather noisy, but after
reconstruction the information is retrieved. Both 3D While there are many benefits of cryo-TEM over
structural approaches (single particle reconstruction more traditional TEM approaches, there are also sev-
and tomography) rely on the Projection Theorem, eral challenges to overcome in order to collect 2D
which states that a Fourier transform of a projection cryo-TEM images with high information content. A
image corresponds to a central plane in reciprocal significant challenge in cryo-TEM is that image con-
space.6,14 Thus, information from multiple projection trast in 2D cryo-micrographs can be quite low.6 Not
images can be combined in reciprocal space to gener- only is the density difference between the specimen
ate a 3D structure. In the single particle reconstruc- and the surrounding frozen water or solvent typically
tion approach, 2D projections of different but low, but frozen specimens are highly beam sensitive
structurally similar particles are combined. In the and they can only tolerate a low electron dose before
cryo-ET approach, 2D projections representing dif- significant damage occurs to the sample. As a general
ferent views of the same sample area are combined. approximation, the electron dose used in cryo-TEM

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 WIREs Nanomedicine and Nanobiotechnology Cryo-electron microscopy and cryo-electron tomography of nanoparticles

is at least a thousand times lower than that used in nanoparticles are often engineered from human
material science TEM applications. In fact, for cryo- viruses, plant viruses, and bacteriophages.63 There
TEM imaging it is best to use low dose software on have been successes with this approach, including
the electron microscope to manage beam exposure to development of vaccines for human papilloma
the selected imaging area. An anti-contamination virus.69 Proteins, such as tumor-specific ligands or
device designed specifically for cryo-TEM is recom- monoclonal antibodies or antigen binding fragments,
mended to prevent water vapor generated during can be linked to nanoparticles to help target particu-
cryo-TEM imaging from re-condensing on the frozen lar cell types via key cellular receptors.70 Liposomes
grid and forming ice contamination. Collection of and nanostructured lipid carriers are well studied
micrographs a few microns out of focus to produce nanoparticles for targeted drug delivery.71–73 They
phase contrast is a standard approach in cryo-TEM have the advantages of biocompatibility, capacity for
to overcome the challenge of low image contrast. self-assembly, and the ability to carry large drug pay-
Distortions in cryo-TEM images, which are caused loads. Polymer based nanoparticles, such as poly-
by out of focus image collection, can be partially meric micelles and dendrimers, are also well studied
corrected by computational contrast transfer func- for drug delivery applications and they can be more
tion (CTF) correction.44–46 Phase plates represent a stable than liposomes.65,74–77 Polymeric nanoparti-
new technological advance that offers the promise cles can be formed with biodegradable polymers,78
of enhanced image contrast for cryo-TEM.47–49 or stimuli sensitive polymers.79 Initial in vitro and
Another challenge faced in cryo-TEM imaging is in vivo safety tests indicate that polymeric nanoparti-
beam-induced motion.50–52 This problem can be cles can be safe for biomedical applications.74 Quan-
overcome by using a direct electron detector, which tum dots, which are colloidal semiconductor
collects multiple sub-second movie frames rather nanocrystals, and polymer dots, which are semicon-
than a single exposure. Software is used to align and ducting polymer nanoparticles, offer brighter fluores-
average the movie frames and produce an enhanced cence emission and longer emission lifetimes
2D image. Direct electron detectors have rapidly compared to fluorescent dyes.67,80 Applications of
revolutionized the cryo-TEM field and are enabling fluorescent nanoparticles include single particle track-
near-atomic resolution structures of biological ing, multicolor imaging, multiplex detection of ana-
macromolecules.53 lytes, and bioimaging.81,82 Metallic nanoparticles
have great potential as biosensors, and for targeted
drug delivery, diagnostic imaging and theranos-
tics.60,68,83,84 Remarkably there are species, such as
TYPES OF NANOPARTICLES the Phoma fungus, that produce anti-microbial nano-
The nanoparticle field has exploded over the past particles containing silver and the field of synthetic
20 years with the design and creation of a wide vari- biology is building upon natural examples such as
ety of particles for applications in medicine, cell and these to create new metallic nanoparticles with
molecular biology, and industry.54–58 By definition, desired functionality.85 Superparamagnetic iron
nanoparticles have dimensions between one to one oxide nanoparticles (SPIONs), which are composed
hundred nanometers. This size range is convenient of iron oxide crystals coated with dextran or another
for biomedical applications, as particles of this size biocompatible compound, have higher magnetic sus-
can travel through the blood stream and can be read- ceptibility than larger iron oxide particles. SPIONS
ily taken up by cells. For drug delivery, nanoparticles become magnetized in the presence of an external
offer the potential of better encapsulation, bioavaila- magnetic field and yet exhibit zero net magnetization
bility, controlled release, and lower toxicity.55,58,59 in the absence of an applied magnetic field. They
For biomedical imaging applications, targeted nano- have applications in both drug delivery and as MRI
particles offer the promise of earlier detection of dis- contrast agents.57,86
ease.60 Nanoparticles are often small enough that As the complexity grows, and as more precise
quantum effects become apparent and this may functionality is strived for, it will become increasingly
impart enhanced optical or magnetic properties to important to structurally characterize nanoparticles.
nanoparticles as compared to bulk materials. Visualizing the 3D structure of nanoparticles should
Nanoparticles may be engineered from biologi- be valuable for understanding how they interact with
cal macromolecules (proteins, DNA, RNA),61–63 the human body in medical applications and with
lipids,64 polymers,65 semiconductors,66,67 metals,68 bulk materials in engineering applications. Evalua-
or a vast number of possible combinations of tion of nanoparticles with 2D cryo-TEM, as well
these components.58 Protein, DNA, and RNA based as 3D cryo-TEM single particle reconstruction and

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cryo-ET, will provide information that is comple- nanoparticles represent a class of lipid-based parti-
mentary to that derived from other characterization cles, which have highly ordered, internal water chan-
techniques including light scattering, SAXS, SANS, nels and which have strong potential for drug
NMR, microfluidic behavior, and computational delivery applications.105 It was shown in the 1960s
studies.87–89 that lipids can self-assemble into non-lamellar phase
structures, including a cubic phase and a hexagonal
phase.106 Dispersed colloidal particles in the cubic
2D CRYO-TEM IMAGES phase are called cubosomes,107,108 and those in the
hexagonal phase are called hexosomes.109 Cryo-
OF NANOPARTICLES TEM has played an important role in visualizing
Icosahedral viruses were among the first specimens and confirming the formation of these nanoparti-
examined by cryo-TEM,1 and numerous cryo-TEM cles.108,109 Beautiful cryo-TEM images of these self-
studies of viruses followed.6,11 Virus-based nanopar- assembled lipid superstructures have been
ticles and virus-like particles (VLPs) that lack the published.110–112 A cryo-TEM image of a hexosome
viral genetic material required for replication are is shown in Figure 1(b) along with a Fourier trans-
potential platforms for vaccine design.63 Simian virus form of a magnified region that confirms the struc-
40 (SV40), which is a polyomavirus found in both tural periodicity within the sample.91 A cryo-TEM
monkeys and humans, can be induced to form differ- study of intermediates in the assembly pathway of
ently sized icosahedral particles depending on the size cubosomes led to a model for their growth pathway
and composition of the nucleic acid component starting with unilamellar vesicles.113 Cryo-TEM has
(DNA or RNA).90 Assembly assays with the SV40 also been used to demonstrate the preservation of
VP1 protein and short (1.9 knt) ssRNA molecules cubosomes after addition of targeting ligands.114
produce 22 nm diameter VLPs that are smaller than A wide variety of polymeric nanoparticles have
mature virions. Cryo-TEM images of the SV40 VLPs been examined by cryo-TEM.9,41–43 Specific recent
reveal homogeneous 22 nm particles, as well as lar- examples include dexamethasone-loaded polymeric
ger more heterogeneous assemblies (Figure 1(a)).90 nanoparticles,115 PEGylated polyester nanocapsules
As this example illustrates, 2D cryo-TEM provides a of perfluorooctyl bromide,116 amphiphilic Janus
direct way to visualize the polydispersity within a dendrimers,117 and polynorbornene amphiphilic
VLP population or nanoparticle preparation with block copolymers.118 Sometimes cryo-TEM image
minimal distortions caused by sample preparation. contrast is low for polymers, particularly if they are
Many cryo-TEM studies have been performed in an extended conformation and are not densely
on lipid based nanoparticles, including liposome, packed. A few tricks have been discovered for
lipopeptide, lipid liquid crystalline, and solid lipid enhancing the contrast of polymers in cryo-TEM
nanoparticles.43,94–104 Lyotropic liquid crystalline images. For example, the addition of cesium ions

(a) (b) (c) (d)

F I G U R E 1 | Examples of cryo-TEM images of protein-RNA based, lipid based, polymeric, and metallic nanoparticles. (a) Protein-RNA
nanoparticles (T = 1 polyomavirus SV40 VP1 virus-like particles) 22 nm in diameters (arrows) and larger assemblies, scale bar, 50 nm. (Reprinted
with permission from Ref 90. Copyright 2013 American Chemical Society) (b) Lipid-based hexosome nanoparticles, scale bar, 50 nm. The lower
panels show a magnified area and a Fourier transform of this area. (Reprinted with permission from Ref 91. Copyright 2005 American Chemical
Society) (c) Spherical polyelectrolyte brushes with added cesium ions and bovine serum albumin to enhance the image contrast of the brush layer,
scale bar, 200 nm. (Reprinted with permission from Ref 92. Copyright 2005 American Chemical Society) (d) Iron oxide polymeric nanoparticles,
40 weight percent iron oxide clustered within the amphiphilic diblock copolymer, 5 K-poly(ethylene oxide)-b-10 K-poly(D,L-lactide), scale bar,
50 nm. (Reprinted with permission from Ref 93. Copyright 2014 American Chemical Society)

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 WIREs Nanomedicine and Nanobiotechnology Cryo-electron microscopy and cryo-electron tomography of nanoparticles

provided some contrast enhancement for spherical biological samples is usually performed with water as
polyelectrolyte brushes, while addition of bovine the vitrified solvent. Polymeric nanoparticles may by
serum albumin adsorbed within the brush layer pro- synthesized in, or undergo self-assembly in, an
vided even more contrast (Figure 1(c)).92 This organic solvent and therefore it may be desirable to
approach enabled the full extent of the polyelectro- image them in a vitrified organic solvent. Cryo-TEM
lyte brush to be visualized by cryo-TEM. The brush with vitrified organic solvents and ionic liquids, as
layer is found to be extended in the osmotic limit well as mixtures of water and organic solvents, has
(no added salt) and nearly collapsed at high ionic been performed.41,42,124–126 Solvents that work well
strength (507 mM added salt). Another trick that has for cryo-TEM include heptane, decane, toluene, iso-
been developed for macromolecules and viruses is octane, and dimethylformamide.127 When the sample
cryo-negative stain.119 This involves adding ammo- is in an aqueous solution, liquid ethane is usually
nium molybdate to the sample before cryo-fixation used as the cryogen for freezing the sample grid.
and it can result in higher signal-to-noise ratios in However, nonpolar solvents are often soluble in eth-
micrographs compared to unstained cryo-TEM. ane so liquid nitrogen may be a better choice for
Metal containing nanoparticles should be emi- the cryogen when preparing grids with vitrified
nently feasible samples for cryo-TEM imaging organic solvents.128 Cryo-TEM imaging in water and
because the density of the metal component is signifi- organic solvents offers a way to examine the mor-
cantly higher than the surrounding vitreous ice. The phological changes that a nanoparticle undergoes
challenge here is that if the nanoparticles also contain when transferred from one solvent to another. For
polymers, these are sometimes poorly visualized by example, mixed poly(acrylic acid) (PAA)/polystyrene
cryo-TEM since polymers typically have much lower (PS) brush-grafted silica nanoparticles have been
density than metals. For example, cryo-TEM images imaged in both water and N,N-dimethylformamide
of metallic nanoparticles formed with 40 weight per- (DMF; Figure 2).124 In this case, uranyl acetate was
cent iron oxide clustered within the amphiphilic added prior to freezing the sample in order to selec-
poly(ethylene oxide)-b-D,L-lactide copolymer, reveal tively stain the PAA chains. Cryo-TEM images of the
high contrast for the metal clusters and a faint nanoparticles showed that the brush layer is more
corona for the surrounding copolymer (Figure 1 compact in water, with a thickness of 18 nm, and
(d)).93 Cryo-TEM images of such nanoparticles can more extended in DMF (27 nm).
be used to quantify the size distributions of the metal
clusters and this can be correlated with the nanopar-
ticle’s relaxation properties for MRI contrast 3D CRYO-TEM STRUCTURES
enhancement. Cryo-TEM has also been used to visu- OF NANOPARTICLES
alize dispersions of colloidal magnetite.120 Cryo-
TEM grids were prepared in presence and absence of Protein Based Nanoparticles
a magnetic field and cryo-micrographs reveal how If a protein based nanoparticle has icosahedral sym-
these magnetite nanoparticles can align with an metry, such as a virus or VLP, it is possible to apply
external magnetic field. Silver nanoparticles cryo-TEM single particle reconstruction methods to
embedded in a biocompatible nanogel have been determine the 3D structure. This cryo-TEM approach
imaged and quantified with cryo-TEM.121 Ferrihy- has yielded atomic and near-atomic resolution virus
drite nanoparticles have been examined by cryo- structures.11 Cryo-TEM single particle reconstruc-
TEM to evaluate their aggregation state in buffers tions have been determined for human papillomavi-
with different ionic strength.122 Mixed-valent iron rus VLPs, both with and without bound antibody
nanoparticles formed electrochemically by electroco- fragments.129 This approach provides visualization
agulation have also been examined by cryo-TEM.123 of the antigenic site on the surface of the nanoparti-
In this case, cryo-TEM images were useful for distin- cle. In another study, cryo-TEM single particle recon-
guishing various nanoparticle aggregates by crystal struction of VLPs formed with the polyomavirus
morphology. SV40 VP1 protein and short (1.9 knt) ssRNA mole-
cules reveals two concentric shells consistent with an
outer protein capsid layer and an internal RNA layer
CRYO-TEM IMAGES IN (Figure 3).90 Volume analysis of the density layers
indicates that a portion of the ssRNA may extrude
ORGANIC SOLVENTS
from the protein capsid.
For biological macromolecules water is the relevant While icosahedral symmetry is convenient, this
medium, and therefore cryo-TEM imaging of high level of symmetry (60-fold) is not essential for

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F I G U R E 2 | Examples of nanoparticle cryo-TEM images in different solvents. (a) Cryo-TEM image of mixed PAA/PS brush-grafted silica
nanoparticles in DMF. The electron dense spots in the center of the image are 10 nm fiducial nanogold markers. (b) One enlarged nanoparticle in
panel (a). (c) Cryo-TEM image of mixed PAA/PS brush-grafted silica nanoparticles in water. (d) One enlarged nanoparticle in panel C. The PAA
chains were positively stained with uranyl acetate. Scale bars, 100 nm. (Reprinted with permission from Ref 124. Copyright 2015 American
Chemical Society).

reconstructed. A requirement for the single particle

reconstruction method is that the sample needs to be
homogeneous, such that projection images of differ-
ent particles can reasonably be presumed to corre-
spond to the same 3D structure. The vault is a
protein-RNA nanoparticle with 39-fold symmetry,
which is almost ubiquitously expressed in eukaryotes
and is being engineered as a functionalized
nanoparticle.132–134 The structures of wild-type and
various engineered forms of the vault have been stud-
F I G U R E 3 | Cryo-TEM single particle reconstruction of a protein- ied by cryo-TEM single particle reconstruction.132–135
RNA nanoparticle with icosahedral symmetry (T = 1 polyomavirus The large size of the vault (71 nm × 42 nm × 42 nm)
SV40 VP1 virus-like particles). Both full (left) and cropped views (right) and its hollow interior make it a promising candidate
are shown in radially colored surface representations. The internal for a drug delivery vehicle. In a recent study, recombi-
RNA shell (blue) appears disconnected from the outer protein shell nant vaults were engineered to encapsulate a phospho-
(red). Oval, triangle, and pentagon symbols indicate the locations of lipid bilayer nanodisk loaded with hydrophobic all-
icosahedral 2-fold, 3-fold, and 5-fold axes, respectively. Scale bar, trans retinoic acid, a potent but toxic therapeutic com-
5 nm. (Reprinted with permission from Ref 90. Copyright 2013 pound.132 Although the vault structure formed by
American Chemical Society). recombinant protein can be determined by cryo-TEM
single particle reconstruction methods, the internal
the cryo-TEM single particle reconstruction approach. nanodisk was not expected to follow the overall sym-
In fact, asymmetric assemblies like the ribosome130 metry of the vault. Therefore, a cryo-electron tomo-
and macromolecules like γ-secretase131 can also be gram was generated of the vault-nanodisk complex.

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 WIREs Nanomedicine and Nanobiotechnology Cryo-electron microscopy and cryo-electron tomography of nanoparticles

Lipid Based Nanoparticles

In the presence of a high concentration of certain
membrane proteins, a lipid bilayer may be induced to
form a polyhedral structure, such as an in the case of
Side an enveloped icosahedral virus.145 Symmetrical mem-
brane protein polyhedral nanoparticles have been
induced to form with two membrane proteins, the
connexion Cx26 and the bacterial membrane pro-
tein, mechanosensitive channel of small conductance
(MscS).146 A cryo-ET study confirmed that mem-
brane protein nanoparticles formed with MscS pos-
sess octahedral symmetry. After confirmation of the
symmetry and structural homogeneity of the sample,
Top it was feasible to determine a cryo-TEM single parti-
cle reconstruction of the nanoparticles. The resulting
cryo-TEM single particle reconstruction was at suffi-
ciently high resolution (0.9 nm) to allow a model to
FI GU RE 4 | Cryo-TEM structure of the vault-nanodisk complex. be built for the inner and outer helices of the MscS
The structure is a composite of a cryo-TEM single particle transmembrane pore.
reconstruction of the vault (green) and a cryo-electron tomogram of Liposomes are generally suitable samples for
the internal phospholipid bilayer nanodisk (red). The overall cryo-ET but not for cryo-TEM single particle recon-
dimensions of the vault are 71 nm × 42 nm × 42 nm. (Reprinted struction because of their inherent heterogeneity in
with permission from Ref 132. Copyright 2011 John Wiley and Sons). size and shape. For example, cryo-TEM images of
Doxil, which is the trade name for a doxorubicin
The cryo-TEM structure shows that the nanodisk liposomal formulation, and which is comprised of
is incorporated within the lumen of the vault pegylated liposomes encapsulating doxorubicin,
(Figure 4).132 reveal a range of diameters for these particles
(Figure 6(a)).147 A sample of this sort may be evalu-
ated in three dimensions by collection of a tilt series
DNA Based Nanoparticles
of images for one region of the specimen on a cryo-
3D cryo-TEM methods have been applied to a
TEM grid. Typically, images are collected over a
variety of DNA based nanoparticles including
range of −70 to +70 . The tilt geometry of the sam-
minicircles,136 self-assembled DNA tetrahedrons,
ple grid, sample holder, and the microscope column
octahedrons, and symmetric polyhedra,137–139 a nan-
prevents collection of a complete −90 to +90 range.
oscale box,140 various 3D DNA-origami objects,141
This results in a so-called ‘missing wedge’ of informa-
nanocages,142,143 and origami frames.144 In a recent
study, 3D cryo-TEM methods were used to evaluate tion in reciprocal space. The effect of the missing
the structure of octahedral DNA-origami frames with wedge is that spherical objects, such as liposomes,
gold nanoparticles attached at all of the vertices or at can appear as cut off on the top and bottom surfaces.
specific vertices.144 Gold nanoparticles were coated It is also possible with specialized microscopes and
with complementary DNA so that they would bind sample holders to collect a double tilt series, with tilt
to ‘sticky ends’ of DNA engineered at the vertices of images taken around two orthogonal axes. This
the DNA-origami frames. The large density differ- results in a smaller missing region region referred to
ence between the DNA and the gold nanoparticles in as the missing cone. Tomograms provides 3D infor-
cryo-TEM necessitated special image processing tech- mation that can be viewed either in density slices
niques in order to produce 3D structures. Single par- (Figure 6(b)), or in a surface representation (Figure 6
ticle reconstructions of the DNA portion of the (c)). Although estimates can be made from 2D cryo-
assemblies were generated from cryo-TEM images in TEM images, a 3D tomogram facilitates more relia-
which the density from the gold nanoparticles was ble measurements of the particle diameters and the
excluded. Reconstructions of the gold nanoparticle size of the encapsulated cargo.147
portion of the assemblies were generated directly Combining liposomes with magnetic nanoparti-
from the high intensity gold nanoparticle signal in cles to form magnetoliposomes is a promising tool
the cryo-TEM images. Composite structures are for drug delivery and treatment of hyperthermia.148
shown in (Figure 5).144 One issue faced in engineering magnetoliposomes is

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(a) (a)

7 nm
(b)

10 nm

F I G U R E 5 | Cryo-TEM single particle reconstructions of gold

nanoparticles built using DNA origami frames. (a) A P6 DNA
octahedron with all sticky ends encoded to coordinate 7 nm
nanoparticles into a symmetric six-nanoparticle cluster. Scale bar,
7 nm. (b) A P4(1234) cluster with 10 nm gold nanoparticles at
(b)
vertices 1-2-3-4 of the octahedral frame. Scale bar, 10 nm. The
structures in panel (a) and (b) are both composites of a reconstruction
of the DNA portion of the origami frame together with a
reconstruction of the gold nanoparticle portion of the assembly. The
composite structures are viewed along the octahedral 4-fold, 3-fold,
and 2-fold symmetry axes (left to right) with the gold nanoparticles
shown in gold and the DNA density radially color coded from red to
blue representing the inner to outer radii. (Reprinted by permission
from Ref 144. Copyright 2015 Macmillan Publishers Ltd: Nature
Nanotechnology).

the inclusion size limitation between the vesicle

bilayers.149 Cryo-TEM images and cryo-ET were (c)
instrumental in confirming that with certain sample
preparation techniques liposome membranes can
accommodate up to 60 nm clusters of SPIONS.149
While 2D cryo-TEM images indicated that the
SPIONS were located between the lipid bilayers, the
3D cryo-electron tomogram confirmed this observa-
tion and also allowed visualization of the packing
arrangement of the SPIONS (Figure 7).149 Other
examples of cryo-ET studies of lipid based nanoparti-
cles include visualization of a silica nanoparticle
engulfed in a liposome,150 and gold particles encap-
sulated within ethasomes, which are vesicles com- F I G U R E 6 | Cryo-TEM analysis of Doxil, a pegylated liposome
posed of phospholipid, ethanol, and water.151 that encapsulates doxorubicin. (a) Cryo-TEM image. (b) Central slice
(0.5 nm thick) from a cryo-electron tomogram. (c) Segmented
representation of a cryo-electron tomogram, with liposome density
Polymer Based Nanoparticles shown in purple and doxorubicin density in pink. Scale bars, 100 nm.
(Reprinted by permission from Ref 147. Copyright 2008 Medicine Ltd).
Several types of polymeric nanoparticles have been
studied by cryo-ET. These include nanowires formed
by the conducting polymer poly(2-hexylthiophene)
(P3HT),125 complexes formed by aggregation of complexity of polymeric nanoparticles can be quite
amphiphilic double-comb diblock copolymers,152 high, as is especially true for multicompartment
and PS nodules on 170 nm silica seeds formed micelles formed with a triblock terpolymer and
under emulsion-polymerization conditions.153 The interpolyelectrolyte complex (IPEC) formation with

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 WIREs Nanomedicine and Nanobiotechnology Cryo-electron microscopy and cryo-electron tomography of nanoparticles

grafted silica nanoparticle.124 Theoretical studies

predicted that in a nonselective good solvent, in
which both types of grafted polymers are soluble,
the polymers should microphase separate laterally.
Also, mixed brush-grafted nanoparticles are environ-
mentally responsive, and morphological differences
are expected for the mixed brush layer in organic
and aqueous solvents. Cryo-ET provides the most
direct way to visualize the in situ morphology of
these nanoparticles. Cryo-TEM images of mixed
PAA/PS brush-grafted nanoparticles in DMF and
FI GU RE 7 | Cryo-electron tomogram of liposomes with water were compared, and revealed that the brush
incorporated clusters of superparamagnetic iron oxide nanoparticles layer is more extended in DMF and more compact
(purple). Scale bar, 50 nm. (Reprinted with permission from Ref 149.
in water (Figure 2). However, 3D analysis with
Copyright 2014 American Chemical Society).
cryo-ET was required to unambiguously distinguish
the difference in brush layer morphology in these
a diblock copolymer.154 Variations of this type of two solvent environments. In DMF, a nonselective
nanoparticle were analyzed by 2D cryo-TEM and good solvent, the mixed brush layer was observed
cryo-ET (Figure 8). The authors make the point to microphase-separate laterally into a ripple struc-
that while the 2D images suggest similar morpholo- ture (Figure 9(a)–(c)). In water, only the PAA poly-
gies for two variants, the 3D cryo tomograms mers are soluble and the PS polymers were found to
reveal otherwise hidden structural features. collapse toward the silica core and form isolated
Another high complexity polymeric nanoparti- surface tethered micelles within a swollen PAA
cle studied by cryo-ET is the mixed PAA/PS brush- brush layer (Figure 9(d)–(f )).

Tilt view of reconstruction

(a) (b) (c) Isosurface model

(d) Side view of top slice (e) (f) Hemisphere of isosurface model

Tilt view of top slice

Top view of middle slice

FI GU RE 8 | Cryo-TEM analysis of a multicompartment micelle formed with a triblock terpolymer and interpolyelectrolyte complex (IPEC)
formation with a diblock copolymer. (a) Cryo-TEM image. (b) Overview of the cryo-electron tomogram. (c) Isosurface representation of the entire
micelle. (d) Side and tilt views of top slice (red). (e) Top view of middle slice (blue). (f ) Isosurface of cropped micelle. Scale bars, 50 nm. (Reprinted
with permission from Ref 154. Copyright 2014 American Chemical Society).

© 2016 Wiley Periodicals, Inc.

 Advanced Review wires.wiley.com/nanomed

F I G U R E 9 | Cryo-TEM analysis of mixed PAA/PS polymer brush-grafted silica nanoparticles in organic and aqueous solvents. (a) One tilt image
from a tilt-series of the nanoparticles in DMF. (b) Central slice from a cryo-electron tomogram of the nanoparticles in DMF. (c) Segmented
representation of one nanoparticle from the tomogram in panel b, with the silica core shown in red and PAA domains in blue. (d) One tilt image
from a tilt-series of the nanoparticles in water. (e) Central slice from a cryo-electron tomogram of the nanoparticles in water. (f ) Segmented
representation of one nanoparticle from the tomogram in panel e, with the silica core shown in gold and PAA domains in green. The PAA chains
were positively stained with uranyl acetate and have a darker contrast. Scale bars, 100 nm in panels (a) and (b), and 50 nm in panels (c–f ).
(Reprinted with permission from Ref 124. Copyright 2015 American Chemical Society).

CONCLUSION out of focus to generate phase contrast in the image.6

In some cases, applying stain to the sample before
In summary, cryo-TEM provides a way to image nano- freezing can be useful for enhancing image con-
particles in a frozen-hydrated state with minimal dis- trast.92,124 Although cryo-TEM was first developed for
tortions to the sample. In cryo-TEM, the sample is biological samples in water, this technique is gaining
flash frozen so there are no drying artifacts. Normally, popularity for polymeric and nanostructures composed
the sample is not stained or metal coated, so what is of hybrid materials.41,42 Cryo-TEM can be applied to
imaged is the actual sample rather than a coated rep- samples in organic solvents or in aqueous-organic mix-
lica. Some of the challenges faced with cryo-TEM are tures. Cryo-TEM offers a way to visualize changes in
high sensitivity of the frozen sample to electron beam nanoparticle morphology in different solvent environ-
damage, beam induced motion of the sample, and rela- ments, as well as changes in nanostructures during
tively low image contrast for samples composed prima- stages of a self-assembly process.
rily of protein, DNA, RNA, lipid, and polymers. As this review seeks to illustrate, cryo-TEM is a
Metallic nanoparticles usually have good image con- powerful way to visualize nanostructures in three
trast in cryo-TEM due to the large density difference dimensions. Cryo-TEM single particle reconstruction
between metal and solvent. The challenge of sample can be applied to nanostructures that have a uniform
sensitivity to electron beam damage can be dealt with and homogeneous structure, such as icosahedral
by using a low electron dose during image acquisition.6 viruses, the vault particle, and DNA-origami frames.
Beam induced motion of the sample can be addressed Moreover, cryo-ET can be applied to nanostructures
by using a direct electron detector with frame aver- that have heterogeneous or irregular structures, such
aging software to correct for motion between sub- as liposomes and polymeric nanoparticles. The differ-
second frames.51,52 To deal with low image contrast, ence in these two approaches is that one involves
typically cryo-TEM images are collected a few microns averaging projection images of multiple nanoparticles

© 2016 Wiley Periodicals, Inc.

 WIREs Nanomedicine and Nanobiotechnology Cryo-electron microscopy and cryo-electron tomography of nanoparticles

captured in different views in cryo-electron micro- to the same nanoparticle, if there is a basic uniform
graphs (single particle reconstruction), and the other structure for a nanoparticle and a heterogeneous or
involves tilting the specimen stage and collecting mul- flexible component that would only be observed in a
tiple projection images of a single specimen area tomogram.132 A 3D cryo-TEM structure can resolve
(tomography). A single particle reconstruction has ambiguity present in 2D projection images. A struc-
the potential of higher resolution since many more ture can also facilitate quantitative measurements
projection images can be averaged. Also, a single par- and volume analyses of separate components within
ticle reconstruction does not suffer from the missing a larger assembly. As the complexity of designed
wedge or missing cone problem of tomography, nanoparticles increases, it will become even more
which lead to limited resolution in one direction. important to visualize the structures that are pro-
However, cryo-ET is the best approach for lipid and duced. For all of these reasons, it is easy to predict
polymer based nanoparticles with pleomorphous that cryo-TEM derived 2D and 3D information will
structures. Sometimes there is a reason to apply both play a role in designing the next generation of nano-
cryo-TEM single particle reconstruction and cryo-ET particles with improved and specialized functionality.

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