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Histochem Cell Biol (2009) 132:253–262
DOI 10.1007/s00418-009-0622-0
123
REVIEW
Toward visualization of nanomachines in their native
cellular environment
Jason Pierson · Musa Sani · Cveta Tomova ·
Susan Godsave · Peter J. Peters
Accepted: 7 July 2009 / Published online: 1 August 2009
© The Author(s) 2009. This article is published with open access at Springerlink.com
Abstract The cellular nanocosm is made up of numerous
types of macromolecular complexes or biological nanoma-
chines. These form functional modules that are organized
into complex subcellular networks. Information on the
ultra-structure of these nanomachines has mainly been
obtained by analyzing isolated structures, using imaging
techniques such as X-ray crystallography, NMR, or single
particle electron microscopy (EM). Yet there is a strong
need to image biological complexes in a native state and
within a cellular environment, in order to gain a better
understanding of their functions. Emerging methods in EM
are now making this goal reachable. Cryo-electron
tomography bypasses the need for conventional Wxatives,
dehydration and stains, so that a close-to-native environment
is retained. As this technique is approaching macromolecu-
lar resolution, it is possible to create maps of individual
macromolecular complexes. X-ray and NMR data can be
‘docked’ or Wtted into the lower resolution particle density
maps to create a macromolecular atlas of the cell under nor-
mal and pathological conditions. The majority of cells,
however, are too thick to be imaged in an intact state and
therefore methods such as ‘high pressure freezing’ with
‘freeze-substitution followed by room temperature plastic
sectioning’ or ‘cryo-sectioning of unperturbed vitreous
fully hydrated samples’ have been introduced for electron
tomography. Here, we review methodological consider-
ations for visualizing nanomachines in a close-to-physio-
logical, cellular context. EM is in a renaissance, and further
innovations and training in this Weld should be fully
supported.
Keywords Cellular nanomachines · Single particle
analysis · Cryo-electron tomography · Vitreous
cryo-sectioning
Introduction
Proteins perform key roles in the majority of cellular func-
tions. They also serve as building blocks for larger
heterogeneous macromolecular assemblies, or biological
nanomachines, which are organized into cellular ‘modules’
(Alberts 1998). As the name implies, a biological machine
is analogous to the man-made version. Both are made of
individual components that function in a coordinated fash-
ion to perform speciWc functions. In nature, if any single
structural component of a nanomachine malfunctions the
result is often a disease. Discerning the ultra-structure of a
biological machine and its components within the cell can
contribute to an understanding of how it functions both nor-
mally, and in disease. Little is known, however, about the
details of how these macromolecular machines are spatially
organized within a cellular environment.
X-ray crystallography and NMR approaches have been
used to complement genetics, biochemistry, and proteomics
to characterize the overall structural organization of indi-
vidual protein complexes. Most macromolecules, however,
cannot be crystallized, or when crystallized, they diVract
poorly, as only large, well-ordered crystals diVract. In the
Robert Feulgen Lecture 2009 presented at the 51st symposium of the
Society for Histochemistry in Stubai, Austria, October 7–10, 2009.
J. Pierson · M. Sani · C. Tomova · S. Godsave · P. J. Peters (&)
Division of Cell Biology,
The Netherlands Cancer Institute - Antoni
van Leeuwenhoek Hospital (NKI-AVL),
Plesmanlaan 121 B6, 1066 CX Amsterdam, The Netherlands
e-mail: p.peters@nki.nl
URL: www.nki.nl/research/peters
254 Histochem Cell Biol (2009) 132:253–262
123
case of NMR, the size of the protein complex is limited to
approximately < 100 kDa (Wider and Wüthrich 1999). In
order to study biological machines where they normally
reside, and not as isolated entities, then more suitable imag-
ing techniques, such as electron microscopy (EM) and elec-
tron tomography (ET), need to be adapted.
Transmission EM is one of the most powerful techniques
for ultra-structural studies of the cell and its constituents
(McIntosh 2001). When performed on sections of Wxed
cells and tissues, it has been invaluable in establishing a
picture of the subcellular arrangement of organelles and
determining the localization of gene products with immuno-
gold labeling techniques. As an example, recently, the route
of tubercle bacillus infection was demonstrated using
immunogold labeling on aldehyde Wxed cryo-sections and
our Wndings revealed a pathway that is against the current
textbook dogma (van der Wel et al. 2007). Nevertheless,
this technique makes use of Wxed, dehydrated and heavy
metal-stained material, and artifacts at the macromolecular
level may be introduced at all stages (Peters and Pierson
2008). Recently, there has been an important focus on opti-
mal sample preservation for high-resolution structural stud-
ies of macromolecules in situ. This quest to Wnd the true
relation between molecules, linked into supramolecular
complexes and assembled into an intricate network of cel-
lular compartments, is revolutionizing modern transmission
EM. The catalyst for this revolution has been cryo-ET
(Dubochet et al. 1988; Lucic et al. 2008). ET is used to gen-
erate 3D maps from a series of 2D transmission EM
images. The specimen is rotated with respect to the imaging
source; in this case, electrons and a series of projection
images are recorded over a limited angular range (usually
¡70° to +70°).
Cryo-ET is based on a freezing technique that captures
the cellular water in an amorphous (glass-like) layer in
which all cellular components are embedded. This process
is known as vitriWcation, which is achieved by the ultra-
rapid freezing of a thin biological sample, circumventing all
harsh chemical Wxatives and heavy metal stain.
Here we analyze diVerent EM imaging procedures that
have the ultimate goal of visualizing macromolecular
‘machines’ in the most natural environment currently possi-
ble. Using a variety of imaging techniques, structural infor-
mation can be extracted at diVerent resolution levels. This
information can be combined to create a multi-resolution
density map of the individual macromolecular complexes;
eventually leading to a catalog of all biological nanomachines
that can be used to search in 3D volumes (Bohm et al.
2000) of cells, and to determine the spatial organization of
individual complexes within a cellular environment (Nickell
et al. 2006). In addition, we give insight on what will be
important for the future to achieve this goal of mapping
macromolecular machines within a cellular context.
Single particle analysis
Electron microscopy has provided important structural
information about protein complexes essential for describ-
ing their functional organization. Single particle analysis
(SPA) has been used to determine the structure of a variety
of macromolecules and biological nanomachines. This
involves the analysis of images from large numbers of puri-
Wed, macromolecular particles that have been placed on an
EM grid. These images are used to build up a 3D model of
the complex.
Sample preparation
For SPA, a convenient isolation approach is to ‘Wsh’ the
nanomachines out from their cellular environment using a
tag that targets a single component of the complex, most
importantly often in a single step (Kelly et al. 2008). Nega-
tive staining is often the Wrst step to check for quality and
suitability. The sample is embedded in heavy metal salts,
such as uranyl acetate or phosphotungstic acid, providing a
contrasting agent for the weakly electron-scattering biologi-
cal molecules. This staining has been part of the most com-
monly used EM preparation methods since its introduction
50 years ago (Brenner and Horne 1959). An example can
be seen in Fig. 1 that shows a negatively stained S. Xexneri
needle appendage. The averaged projection view shows the
basal part of the needle complex, composed of several rings
that traverse the cell envelope. The heavy metal stain can,
however, create a cast around the surface of the specimen.
Moreover, dehydration of the specimen can potentially alter
Wne structural details. It also imposes a resolution limit of
10–20 Å (Amos et al. 1982; Sani et al. 2007). These limita-
tions have been overcome by cryo-Wxation, or embedding
of the specimen in vitreous water (Dubochet et al. 1988).
Image analysis
Irrespective of the sample preparation method, individual
particles must be aligned and averaged to improve the signal.
For cryo-preparation methods the averaged particle can con-
tain information to atomic resolutions (Henderson 1995; van
Heel et al. 2000), but suVer from limited contrast. The imag-
ing electron dose is limited (low-dose imaging) to 10–20
electrons/Å2 (Glaeser and Taylor 1978; Dubochet et al.
1988) to reduce the number of inelastically scattered elec-
trons (Stark et al. 1996) that often lead to radiation damage.
To overcome this problem, single particle image enhance-
ment programs have been developed (van Heel et al. 1996;
Frank et al. 1996) to average large numbers of copies of sin-
gle particles and improve the signal-to-noise ratio.
The Wrst step in the averaging procedure is to identify
individual particles within the micrograph manually or
Histochem Cell Biol (2009) 132:253–262 255
123
by using semi-automated computer programs. Particle
alignment then modiWes the rotational and translational
position of each particle, such that all particles have a
comparable orientation and key features will become
visible. Since the resolution depends on the accuracy of
the particle alignment, there must be suYcient particle
feature recognition in a noisy image. Larger complexes
are easier to align and subsequently process. A mini-
mum particle size of 1,000 kDa is therefore desired for
proper alignment (Henderson 1995). The more particles
used in the reconstruction, the higher the resolution of
the Wnal model. The technique is most powerful when
the particles display intrinsic icosahedral (Böttcher et al.
1997), helical (Miyazawa et al. 2003), or crystalline
(Henderson et al. 1990) symmetry, that in certain
instances such as polyhedrosis virus (Yu et al. 2008),
aquaporin-1 (Murata et al. 2000; Engel 2003) and of the
bacterial Xagellum (Yonekura et al. 2003) have reached
atomic resolution.
Asymmetric complexes continue to remain challenging
to images using SPA. The ribosome, which is one of the
most analyzed low symmetry single particles, is an excep-
tion. It has served for many years as test specimen in the
development of many single particle techniques (Stark
2002). Although the resolution is lower for such asymmet-
ric particles, a quantitative description, on the quaternary
level, is nevertheless possible.
Visualizing nanomachines in a cellular context
A drawback with the single particle technique is that the
nanomachines may undergo changes during isolation,
such as loss of labile components, or certain conforma-
tional states may be favored. Furthermore, there are many
macromolecular assemblies associated with membranes,
which should be studied without extraction, in a cellular
context.
Specimen preservation is one of the most critical steps in
the entire process of native cellular imaging. Cellular water
in liquid form is incompatible with the vacuum of the EM.
Water is the most abundant cellular constituent and there-
fore important for preserving cellular ultra-structure. Cur-
rently the only way to Wx cellular constituents without
introducing signiWcant structural alterations is by cryo-
Wxation. To be successful, the freezing process has to be
ultra-rapid in order to avoid ice crystal formation, which
damages cellular material and can hinder imaging. There
are currently two common methods employed; plunge
freezing into liquid ethane (Dubochet et al. 1988) and high
pressure freezing (HPF, Studer et al. 2001).
Cryo-Wxation
The plunge freezing technique is suitable for samples with
limited thickness [i.e., bacteria, isolated cellular organelles
Fig. 1 From a whole cell to isolated complexes. a Electron micro-
graphs of osmotically shocked S. Xexneri exhibiting the type III secre-
tion system protruding through the bacterial envelope (see boxed
area). b Gel chromatography of solubilized secretion complexes from
the bacterial envelope. c The fraction containing enriched complex is
checked by SDS and individual bands identiWed by mass spectrometry.
d 2D projection average of 1,500 isolated needle complexes composed
of a hollow needle appendage (indicated by a stain-penetrated line
along its axis) and a basal part of several rings that traverse the cell
envelope. The channel that runs through the needle appendage is 2–
3 nm in diameter indicating that substrates that exit the conduit do so
in an unfolded state
256 Histochem Cell Biol (2009) 132:253–262
123
(Nicastro et al. 2006) or viruses] or relatively thin regions
of larger cells (Medalia et al. 2002). The physical properties
of water, namely its poor heat conductivity, are the reason
that freezing is limited for large cells and tissues. Currently,
the only method to vitrify thicker samples (up to 200 m) is
by HPF.
The high pressure introduced at the moment of freezing
lowers the freezing point of water. Synchronized pressuri-
zation and cooling of the sample takes place within 20 ms
(Studer et al. 2008). The development of this technique has
allowed samples of various thicknesses to be vitriWed,
although vitriWcation of most mammalian cells in the
absence of non-isotonic cryo-protectants has not yet been
established. Cryo-Wxation has two distinct advantages over
chemical Wxation. It is achieved within milliseconds and it
ensures simultaneous immobilization of all macromolecular
components. Many protein networks are very labile and fall
apart with the slightest osmotic or temperature change and
these unwanted eVects are minimized during cryo-Wxation.
These techniques allow the study of biological samples
with improved ultra-structural preservation, and can facilitate
the study of dynamic processes.
Freeze-substitution
Achieving optimal preservation of the biological sample is
undoubtedly critical, but still only the Wrst step in visualiz-
ing it. Until it was demonstrated that vitreous water could
be sectioned and cellular structure visualized directly in the
cryo-EM (Dubochet et al. 1988), the only way of examin-
ing the cryo-Wxed sample was by embedding it in resin for
sectioning. For this the vitriWed water in the HPF frozen
specimen must be replaced by an organic solvent in a pro-
cess known as freeze-substitution (FS).
Freeze-substitution is a process of dehydration, per-
formed at temperatures low enough to avoid the formation
of ice crystals and to circumvent the damaging eVects
observed after ambient-temperature dehydration. Aggrega-
tion of macromolecules in organic solvents and changes of
the hydration shell surrounding the biological molecules
can occur even at very low temperatures, but it is reason-
able to assume that FS at temperatures below a speciWc
threshold preserves the hydration shell (Hobot et al. 1985;
Kellenberger 1991). Further, the total or partial loss of the
hydration shell can be reduced during the cryo-embedding
procedure, so minimizing aggregation and the redistribu-
tion of diVusible elements (Edelman 1991; Quintana et al.
1991).
Freeze-substitution combines instant physical immobi-
lization of the cell constituents and resin embedding. Once
substitution is complete, samples are gradually warmed-up
and processed further as for conventionally prepared samples.
Successful cryo-Wxation followed by FS shows superior
preservation of Wne structure compared to chemical Wxa-
tion techniques (Müller 1992; Steinbrecht and Müller
1987). This technique also gives the possibility of examin-
ing thick (200–300 nm sections) samples by ET, so that
relatively large cellular volumes can be studied in 3D. This
approach is very beneWcial for an understanding of the
complex relation between diVerent cellular organelles and
randomly occurring events. Membrane contact sites
between the ER and the outer-most membrane of the api-
coplast (a plastid found in most parasites from Apicom-
plexa Toxoplasma gondii and Plasmodium species) can be
seen in Fig. 2 as an example of a sample that has been
freeze-substituted in 0.1% uranyl and embedded in Lowic-
ryl HM20 (Tomova et al. 2009). In addition, this image
shows the potential that HPF/FS has in combining optimal
structural preservation with protein detection and for
applying ET to thick sections. There are numerous exam-
ples where HPF/FS combined with ET have changed our
understanding of cellular structure and dynamics (Marsh
et al. 2001; Murk et al. 2003; Perkins et al. 2001; Perktold
et al. 2007; Tomova et al. 2006; Zeuschner et al. 2005).
Recently, it was shown that cellular Wbrils connect
protoWlaments directly to the inner kinetochore (McIntosh
et al. 2008), which nicely illustrates a mechanism for
directly harnessing microtubule dynamics for chromosome
movement.
Even though numerous diVerent substitution protocols
exist, there are only a few methodological investigations
concerning the process of FS, such as the replacement of
ice by an organic solvent at temperatures of ¡90 to 0°C
(Müller et al. 1980), the substitution capacities of diVerent
organic solvents in the presence of water (Humbel and
Müller 1986), and the inXuence of the diVerent solvents on
the ultra-structural preservation of high-pressure frozen,
freeze-substituted samples (Studer et al. 1995). It is still
arguable what alterations of the cellular structure are
induced and to what extent the information obtained can
be considered as an accurate representation of the living
cell, especially in the case of the 3D structure of macro-
molecules.
Vitreous cryo-sectioning
The expansion and development of cryo-ET and cryo-sec-
tioning of native vitreous samples such as yeast and
bacteria has made it possible to visualize cells directly in
the absence of chemicals, heavy metals and hypertonic
cryo-protectants. Vitreous cryo-sections of Saccharomyces
cerevisiae (Fig. 3) showed that the nuclear envelope forms
a unique membrane contact site with the vacuole, called
the nucleus–vacuole (NV) junction (Millen et al. 2008).
‘Velcro’-like protein–protein interactions hold the junction
in place. The proteins involved are Vac8 in the vacuole
Histochem Cell Biol (2009) 132:253–262 257
123
membrane, which is associated with Nvj1 in the outer
nuclear membrane. NV junctions are the site of piecemeal
microautophagy of the nucleus, which is a type of autophagy
that targets portions of the nucleus.
Artifacts induced by the process of sectioning (knife
marks, crevasses, and compression) have been described in
detail and must be taken into consideration (Al-Amoudi
et al. 2005). An example of a crevasse is shown in Fig. 3b,
where crevasses are partially visible in the vacuole (V) as
white stripes perpendicular to the cutting direction (parallel
to the NV junction). In certain cases crevasses can penetrate
into the depths of the cryo-section of a vitreous sample and
aVect the underlying biological ultra-structure, however, in
ultra-thin sections (< 50 nm) these artifacts appear to be
non-intrusive to the biological ultra-structure.
It is possible to minimize knife marks and crevasses,
making compression the most problematic artifact. Com-
pression is a deformation that makes the vitreous section
shorter along the cutting direction without changing the
overall volume. The result is an increase in overall section
thickness. Thirty percent compression within vitreous cryo-
sections is not unusual. If compression is evenly distributed,
then a reverse transform (Al-Amoudi et al. 2005) can be
applied to the entire volume post-reconstruction. However we
Fig. 2 High pressure freezing/FS combined with ET reveals the rela-
tion between cellular compartments. Images illustrating the relation
between the ER and the apicoplast (a plastid acquired by secondary
endosymbiosis) outermost membrane in the parasite Toxoplasma gon-
dii. a Image from the aligned tilt stack acquired from a thick section
(200 nm), at ¡35° tilt. The gold particles, used for also for the align-
ment of the series, are speciWc labeling for the apicoplast speciWc Acyl
carrier protein. The relation between the membranes of the ER and the
apicoplast is pointed (black arrowhead). b Optical slice from the tomo-
graphic reconstruction of the same apicoplast. The lamellar structure o
f
all the membranes is visible. No additional contrasting was applied.
The close alignment of the outermost apicoplast membrane and the ER
is pointed (black arrowhead). c View of a model generated by the
tomographic reconstructions sown in b. Additional observation in
these images is the budding of vesicles from the nuclear envelope
(white arrowhead). Sample substituted in 0.1% uranyl and embedded
in Lowicryl HM20. Sections were not post-stained. The scale bars in a
and b are 100 nm
Fig. 3 Tomography of thin vitreous cryo-sections of the Saccharomy-
ces cerevisiae nucleus–vacuole ju nction. a A 10-nm slice from a recon-
struction that shows the consistent width of the nuclear membranes in
relation to the vacuole. ‘N’ denotes the nucleus and ‘V’ the vacuole.
Scale bars 50 nm. b A second reconstruction that shows more of an
overview of the region of the NV junction. The inner and outer mem-
branes of the bulk nuclear envelope (white arrows) are further apart
compared to those of the nuclear envelope at the NV junction. Aroun
d
and within the nucleus, macromolecular complexes can be observed,
along with a mitochondrion at the bottom of the image
258 Histochem Cell Biol (2009) 132:253–262
123
have seen indications that compression is rather non-homo-
geneous in Wlament bundles (Salje et al. 2009), microtubules
(Bouchet-Marquis et al. 2007), desmosomes (Al-Amoudi
et al. 2007), and bacterial chemotaxis receptors (Zhang
et al. 2004). On the nanomachine level the aVect of these
artifacts is uncertain, but could be minimal for rigid, macro-
molecular machinery (J. Pierson et al., unpublished).
In addition, extreme caution must be taken during image
acquisition. Beam induced alterations in the sample are
well-known to be a problem, and are often believed to be
the rate limiting step for achieving optimal cryo-EM. It is
no secret that vitreous sections ‘Xow’ during image acquisi-
tion (Sartori Blanc et al. 1998; Sartori, et al. 1996), which is
considered by some researchers to be due to local charge
accumulation and/or radiochemical processes. Therefore, it
is important to ‘learn’ how to read vitreous sections with
respect to structural integrity and preservation (Dubochet
et al. 2007). A common practice is to acquire a single pro-
jection image, using a very low electron dose, before a tilt
series is collected, and then compare it to an image taken
after the series is complete. If there is no noticeable change,
then the structural integrity of the vitreous section has been
maintained. If the Wnal image appears ‘smooth’ (with no
knife marks and/or crevasses visible) then the ultra-struc-
ture of the cell must be analyzed with caution. Map mon-
taging helps to reduce the ‘overhead’ electrons used to
pinpoint an area of interest before acquiring a tilt series. In
order to preserve the vitreous section integrity, the electron
beam dose is restricted, which in turn limits the contrast
levels and the signal-to-noise ratio. This makes subsequent
analysis of such images rather diYcult.
The application of tomography of vitreous sections is
the future of cellular imaging, combining close-to-native
preparation techniques with a 3D view of the vitreous sec-
tion. The ultimate goal is to map complex macromolecular
machines within 3D slices of large cells and tissue. An
example is shown of a sample of Saccharomyces cerevisiae
that was immobilized in a copper tube using HPF. The sam-
ple was vitreous cryo-sectioned into thin (50 nm) slices and
observed using cryo-ET (Fig. 4).
Fig. 4 A diagram illustrating the vitreous cryo-sectioning technique.
a A confocal laser image of Saccharomyces cerevisiae (Dr Maxim
Zakhartsev and Doris Petroi, International University Bremen, Ger-
many). The cells are rapidly frozen by high pressure freezing (HPF) in
copper tubes and then sliced (b) into very thin (50 nm) vitreous cryo-
sections. b A relatively long ribbon of vitreous cryosections is held by
the operator and pulled away from the diamond knife. The ribbon is
placed on an EM grid and imaged by Cryo-EM and -ET. c A medium
magniWcation single image showing a 50 nm vitreous slice of a single
Saccharomyces cerevisiae cell. Scale bar 2 m. In the corner of the
image are holes within the carbon support Wlm (white round circles).
Subsequent cryo-ET is shown in d, e. d A single 2D projection image
from a tilt series. It is diYcult to interpret such images due to the
projection problem of overlapping structures, along with a low signal-
to-noise ratio and contrast levels. Scale bar 100 nm. e Multiple single
projection images can be aligned and reconstructed into a 3D volume
(in this case using IMOD (Kremer et al. 1996)). A 5-nm slice from a
reconstructed volume. In this image the cellular environment can be
visualized much better than in the single projection image (d). The
inset displays a selected area (orange-bounded box) from the tomo-
graphic reconstruction (e)
Histochem Cell Biol (2009) 132:253–262 259
123
Within the volume of the reconstruction, individual
macromolecular complexes can be observed (Fig. 5).
Based on their size and distribution we assume that these
are native 80S ribosomes in various conformational
states, which can also be associated with a variety of pro-
tein complexes that aid in protein translation. The true
identity, however, remains subjective until a suitable
labeling or correlative approach is developed for the vitre-
ous cryo-sectioning.
The future of charting nanomachinery in a native
cellular context
We have witnessed light microscopy breaking the diVrac-
tion barrier, and in the future the spatial resolution will only
improve, with the development of super-resolution light
microscopes. Recently it became clear that the green Xuo-
rescent protein (GFP) tag has a 10–50 times better signal-
to-noise ratio at cryogenic conditions because of reduced
photo-bleaching (Sartori et al. 2007). In combination with
EM, correlative cryo-LM–EM oVers a promising opportu-
nity to investigate cellular organization (Sartori et al. 2007;
Schwartz et al. 2007; Plitzko et al. 2009).
Rather than using a single clonable Xuorescent (GFP)
tagged construct widely used in light microsocopy, a
heavier circular string of Wve GFP’s in tandem could be
constructed that would be visible in the rather noisy and
low contrast cryo-ET images. In addition, the GFP could be
coupled to ferritin or another analog electron dense metal-
lothionein molecule to provide a label for cryo approaches
(Mercogliano and DeRosier 2007).
High-resolution structures produced by X-ray crystal-
lography or NMR can be Wtted or docked into lower
resolution EM images to create a multi-resolution map of
the protein structure. Such bridging techniques will be
instrumental in providing a more complete understanding
of the structure and function of molecular complexes than
is possible with a stand-alone technique.
Recently, volumetric averaging, which is based on the
single particle averaging techniques presented, was applied
to isolated organelles (Nicastro et al. 2006) and to cellular
protein complexes involving cadherins in epidermal des-
mosomes of native skin (Al-Amoudi et al. 2007). In the lat-
ter case, a high-resolution X-ray crystal structure was
placed into the lower resolution cadherin EM map, which
resulted in a multi-resolution map of the cadherin in native
epidermal desmosomes.
In our own lab, one aim is to further our knowledge on
the mechanism of phagocytosis of Mycobacterium tubercu-
losis in human cells. Contrary to the previously accepted
view, we have shown using cryo-immunogold EM that the
mycobacteria can translocate from the conWnes of the
phagosomal membranes into the cytosol, where they repli-
cate more rapidly and cause cell death (van der Wel et al.
2007). We are now using SPA to investigate the type VII
secretion system in isolated bacteria and ultimately we aim
to image mycobacteria in sections of HPF frozen human
cells, and to gain an understanding of the mechanism for
type VII-mediated translocation. The SPA, X-ray and NMR
data can then be docked into the images from vitreous sec-
tions to construct a macromolecular map of the tubercle
bacillus within the host cell.
‘Cutting edge’ technology
The only way to look inside large cells and tissue, at a
close-to-native preservation, is by HPF and vitreous sec-
tioning for cryo-EM. The diamond knife and accessory
tools used for cryo-sectioning are improving, which will
tremendously improve both the quality of the vitreous sec-
tions and subsequently the quality of the imaging. We fore-
see that in the near future we will use robotics for section
manipulation to make handling even more convenient. An
alternative approach to mechanical sectioning is based on
focused ion beam milling (Marko et al. 2007). A futuristic
‘nano-knife’ has also been proposed (Singh et al. 2009)
constructed from multi-walled carbon nanotubes. In the
future these techniques may be able to reduce the distor-
tions currently caused by mechanical vitreous sectioning
with a diamond knife, but at the current state are rather
technically demanding and in development.
Fig. 5 Charting macromolecular machines within a vitreous cryo-sec-
tion. a A selected area (orange-bounded box in Fig. 4d) from a tomo-
graphic reconstruction. b, c The volume has been surface rendered in
order to visualize the cellular organization of macromolecular com-
plexes within a thin vitreous cryo-section from a whole Saccharomyces
cerevisiae cell
260 Histochem Cell Biol (2009) 132:253–262
123
The future of cryo-electron tomography
The current, rather conservative, approximation of the
resolution limit of 4–5 nm is expected to improve as the
need for high-resolution structural information steadily
increases. The electron source is a suitable starting point
for improvement. We have witnessed a dramatic increase
in spatial coherence of electrons using a Weld emission
electron gun (FEG) rather than a LaB6 or tungsten Wla-
ment. As the FEG improves, the induced specimen dam-
age will be further minimized and the resolution may
improve.
Contrast is generated diVerently with unWxed, unstained
material. The objective lens is defocused creating phase
contrast. By defocusing the objective lens, the contrast
transfer function is altered and for high resolution studies
it will need to be accurately assessed through the volume
of single projection images, which is not trivial compared
to single projection images. A comparison between CTF
corrected and uncorrected can be seen in chromosome
arrangement within mitotic HeLa cell sections (Eltsov
et al. 2008).
Electron detectors for image collection are also becom-
ing more sophisticated. The problem of electrons backscat-
tering from the Wberoptic plate into the scintillating layer of
a charge couple device in current detectors can be partially
solved by direct detection of electrons (a complementary
metal-oxide-semiconductor) rather than relying on a scintil-
lating layer (Jin et al. 2008). Remote microscopy control
allows users from around the world to operate and control
microscopes in a completely diVerent part of world. We
have seen that the loading of cryo samples has now become
completely automatic thanks to the cryo-autoloader, which
is an integral part of the Titan Krios (FEI Company). One
million particles taken from several EM grids under cryo-
genic conditions can be imaged and processed automati-
cally in 1 week (H. Stark, personal communication). This
trend should not be limited to imaging but also to the sam-
ple preparation.
In the past two decades, scientiWc challenges in physics,
chemistry, and molecular cell biology have moved toward
the molecular, nanometer-scale domain. Nanoscience, once
only a discipline of theoretical subjects, has now been
established as a vibrant multidisciplinary area of research
and development.
For EM labs to participate fully in this Weld, it will be
necessary to rationalize EM resources. In the Netherlands
for example, about 25 EMs are dispersed throughout the
country. Some have old instruments and low scientiWc
impact, while their maintenance (service) costs are high.
None have the resources needed to initiate a Dutch EM cen-
ter as part of the much-needed network of major European
centers outlined in the strategic roadmap of the European
Strategy Forum for Research Infrastructure (ESFRI). A pre-
requisite to realizing these potential centers, however, is a
source of funds for such a major facility and to merge
resources and personal. National cryo-EM centers will be
optimally suited to become centers of excellence in speciWc
areas of molecular life science and health and there are sci-
entists in many countries with excellent track records in
their respective EM-related Welds. With the combined
eVort, expertise and training as was shown within the EU
3D-EM network, several centers should emerge with bril-
liant junior and senior principle investigators that should
have the right scientiWc setting for acquiring suYcient
research grants to make it self sustainable with the help of
bright young scientists. We are working toward generating
such a center. For more information please visit http://
www.necen.nl.
Summary
The cell is composed of a highly organized network of
macromolecular machines addressing the demanding
requirements of a living cell. Imaging these ‘nanoma-
chines’ in a cellular, close-to-native state is not a trivial
task. Isolation techniques will continue to be essential in
solving the structures of these types of protein complexes
by the ‘divide and conquer’ method (McIntosh 2007). For
analysis of these nanomachines in their normal environ-
ment, rapidly freezing the cell, and cellular elements, in a
vitreous layer of water is currently the only technique that
retains a physiological state of the organism or cells before
EM imaging. As we have seen (Nicastro et al. 2006),
unmatched structural information can be obtained. Using
vitreous cryo-sections, Cryo-ET can be extended to any cell
and almost all tissue. Yet, one major limitation is the proper
vitriWcation under physiological conditions and the locali-
zation of each macromolecular complex, along with their
binding partners, throughout the thickness of the vitreous
cryo-section. As the Weld of cellular imaging of nanoma-
chines continues to evolve, we are certain to gain invalu-
able cell biological insights. We are also likely to see many
nanotechnology applications that utilize these elegant cellu-
lar machines.
Acknowledgments We thank Shoaib Amini for assistance with
Figs. 4and 5. We also thank members of the Peters lab and members
of the Netherlands Centre for Nanoscopy (http://www.necen.nl) and
Helmut Gnaegi from Diatome for stimulating discussions. This work
was performed with support oV the Sixth Research Framework Pro-
gram of the European Union, Project ImmunoPrion (Food-023144),
the NIMIC consortium (Nano-IMaging under Industrial Conditions
http://www.realnano.nl) and Aeras Global TB Vaccine Foundation
which has received a grant for this project from the Netherlands Direc-
torate-General of Development Cooperation (DGIS) Dutch Ministry of
Foreign AVairs.
Histochem Cell Biol (2009) 132:253–262 261
123
Open Access This article is distributed under the terms of the Crea-
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noncommercial use, distribution, and reproduction in any medium,
provided the original author(s) and source are credited.
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