Commentary
7
Visualizing cellular processes at the molecular level
by cryo-electron tomography
Kfir Ben-Harush1, Tal Maimon1, Israel Patla1, Elizabeth Villa2 and Ohad Medalia1,*
1
Department of Life Sciences and the National Institute for Biotechnology in the Negev, Ben-Gurion University, Beer-Sheva, 84105 Israel
Max-Planck-Institute for Biochemistry, D-82152 Martinsried, Germany
2
*Author for correspondence ([email protected])
Journal of Cell Science 123, 7-12 Published by The Company of Biologists 2010
doi:10.1242/jcs.060111
Journal of Cell Science
Summary
The cellular landscape rapidly changes throughout the biological processes that transpire within a cell. For example, the cytoskeleton
is remodeled within fractions of a second. Therefore, reliable structural analysis of the cell requires approaches that allow for instantaneous
arrest of functional states of a given process while offering the best possible preservation of the delicate cellular structure. Electron
tomography of vitrified but otherwise unaltered cells (cryo-ET) has proven to be the method of choice for three-dimensional (3D)
reconstruction of cellular architecture at a resolution of 4-6 nm. Through the use of cryo-ET, the 3D organization of macromolecular
complexes and organelles can be studied in their native environment in the cell. In this Commentary, we focus on the application of
cryo-ET to study eukaryotic cells – in particular, the cytoskeletal-driven processes that are involved in cell movements, filopodia
protrusion and viral entry. Finally, we demonstrate the potential of cryo-ET to determine structures of macromolecular complexes in
situ, such as the nuclear pore complex.
Key words: Actin, Cytoskeleton, Cryo-electron tomography, Herpes simplex virus, Nuclear envelope
Introduction
Cellular activities rely on the concerted actions of macromolecular
complexes that function within dynamic networks. For instance,
the cell cytoskeleton is remodeled within fractions of a second, thus
modulating cell shape and function. Consequently, at the molecular
level, cellular architecture rapidly changes during the cell cycle and
throughout various biological processes. Despite the wealth of
information that exists on cellular components and their dynamic
properties, our current understanding of the functional interactions
that lead to a given cellular process is rather limited. Developing
the experimental tools necessary for the analysis of complex and
variable supramolecular structures inside cells is crucial to rectify
the situation. Microscopical imaging techniques, which now offer
resolution at the previously unattainable nanometer scale, would
thus be expected to provide novel insight into the local and global
organization of functional modules and networks inside cells
(Robinson et al., 2007).
Microscopy techniques have traditionally been central in driving
cell biology forward. Fluorescence microscopy and confocal laserscanning microscopy revolutionized our thinking and opened up a
large set of possible strategies for investigating cellular processes
(reviewed by Schliwa, 2002). In particular, the introduction of green
fluorescent protein (GFP) and its analogs has allowed kinetic
measurement of proteins in living cells (Tsien, 1998). Additionally,
the development of ultra-high-resolution fluorescence microscopy,
such as PALM (Betzig et al., 2006), STED (Hell, 2003) and structured
illumination (Schermelleh et al., 2008), allowed the visualization of
individual macromolecular complexes such as the nuclear pore
complex (NPC) (Schermelleh et al., 2008). However, such approaches
can focus on only a limited number of proteins at a time, depending
on the number of fluorophores available. The molecular architecture
can, however, be reconstructed in three dimensions and at high
resolution by electron microscopy (EM), particularly cryo-electron
tomography (cryo-ET), therefore complementing fluorescencemicroscopy techniques.
In this Commentary, we focus on the principles and implementation
of cryo-ET in the field of cell biology. We demonstrate the
possibility of resolving three-dimensional (3D) cytoskeleton networks
within intact cells by showing actin cytoskeleton networks in the
native context of membranes, vesicles and other molecular complexes.
The potential of using cryo-ET for following viruses as they infect
cells will be shown and discussed. We also consider how the
application of cryo-ET to visualize intact nuclei, in combination with
3D-averaging procedures, has yielded a 3D structure of active NPCs.
Finally, we consider future prospects for cryo-ET. We focus on the
application of this technique to eukaryotic cells; however, it should
be noted that cryo-ET has been successfully used for the study of
prokaryotes and viruses (Borgnia et al., 2008; Briggs et al., 2006;
Gan et al., 2008; Grunewald et al., 2003; Kurner et al., 2005; Lieber
et al., 2009; Liu et al., 2008; Morris and Jensen, 2008).
Cryo-ET: technical notes
Uniquely among imaging techniques, cryo-ET can generate 3D
information concerning the macromolecular architecture of cells in
an unperturbed state (Cyrklaff et al., 2007; Li et al., 2007; Nickell
et al., 2006). Using this technique, one can depict unique cellular
states and reconstruct molecular networks. Through vitrification by
rapid freezing, biological material can be physically fixed, ensuring
close-to-life conditions in samples prepared for cryo-ET (Dubochet
et al., 1988). Because neither chemical fixation nor staining is
needed, the delicate cellular landscape is preserved during sample
preparation and accurately depicts in vivo conditions.
In cryo-ET, 3D structures of specimens are retrieved from 2D
micrographs. Owing to the large depth of focus, electron
micrographs are essentially two-dimensional (2D) projections of a
3D object in the direction of the electron beam. Consequently,
features of the sample are superimposed and cannot be separated,
in contrast to the situation in confocal laser-scanning microscopy.
Nevertheless, the three-dimensionality of an object can be retrieved
by recording a series of projections at varying angles and
Journal of Cell Science
8
Journal of Cell Science 123 (1)
synthesizing these projections into a 3D density map – that is, a
tomogram (Fig. 1) (Frank, 1992). In practice, the different projection
images are collected by tilting the specimen incrementally around
a single axis inside the electron microscope that is perpendicular
to the optical axis of the electron beam (Fig. 1A). This tilt series
is then aligned to a common frame of reference, followed by
calculation of the tomogram (Fig. 1A), most commonly by using
a ‘weighted back-projection’ algorithm (Radermacher, 1992).
The resolution of a tomogram is directly dependent on the angular
increment between two adjacent projections and on the total number
of images that are obtained (Koster et al., 1997). Therefore, the aim
is to collect as many tilted projections as possible, covering the
widest possible angular range, but keeping the electron dose at a
sub-critical level. Thus, the cumulative electron dose in the entire
tilt series must be kept within tolerable limits, typically not
exceeding ~6000 e–/nm2, to prevent radiation damage to the
biological specimen. Furthermore, because of technical limitations,
the tilt series cannot cover the entire spectrum of views and is limited
to ±70°. In practice, a typical tilt series consists of 80-100 exposures
and covers only 120°-140° of the 180° angular range. Consequently,
elongation of features along the beam axis is evident because of a
missing ‘wedge’ in the 3D Fourier space (Frank et al., 2002).
Overall, to minimize the exposure time and to increase the accuracy
of the process, data acquisition must be fully automated, and relies
on computer control (Dierksen et al., 1993; Dierksen et al., 1992).
A further limitation is that the application of cryo-ET to
eukaryotic cells is restricted to relatively thin regions. When the
object is thicker than the mean free path of an electron [200 nm
and 350 nm for 120 keV and 300 keV (acceleration voltage of
electrons in the electron microscope), respectively (Grimm et al.,
1998)] multiple scattering events substantially degrade the image
quality, despite the use of high to medium acceleration voltage
(300 keV) and an energy filter to minimize this effect (Grimm
et al., 1996). As a consequence, samples thicker than 1 m can
barely be studied in toto, and require cryosectioning before they
can be subjected to tomographic analysis. Several laboratories have
devoted substantial effort to establishing freeze-hydrated
cryosectioning procedures. The feasibility of this approach has been
shown using cryosectioned rat liver cells, mouse epidermis, and
human epidermis and cardiomyocytes (Al-Amoudi et al., 2004;
Al-Amoudi et al., 2007; Castano-Diez et al., 2007; Gruska et al.,
2008; Hsieh et al., 2002; Salje et al., 2009), but it is still technically
demanding and often gives rise to sectioning artifacts (Al-Amoudi
et al., 2005).
3D visualization of cytoskeletal networks
For decades, EM of actin cytoskeletons was performed on sections
of chemically fixed, detergent-extracted cells shadowed with metal
(Brown et al., 1976; Svitkina et al., 1997), or by using adherent
cells, which have an apical surface that has been mechanically
removed, for EM analysis (Hartwig et al., 1989; Heuser and
Kirschner, 1980). Although these methods provided important
insight into the architecture of actin networks (Small et al., 1994),
the spatial resolution of the structures revealed by these
methodologies was limited, especially in the third dimension. For
instance, connections between actin filaments were difficult to
resolve in detail after metal decoration or replica formation.
Moreover, distortion of the actin network by detergent treatment
does not permit the study of anchorage of actin filaments to
membranes. Most importantly, by means of total internal reflection
fluorescence (TIRF) microscopy using specific probes for actin
nucleation, growth and branching (e.g. Arp2/3) it was shown that,
Fig. 1. Applying cryo-ET to eukaryotic cells. (A)2D
projections at different tilt angles for individual 3D objects,
such as an intact eukaryotic cell, are recorded by tilting the
specimen holder; the projections typically cover ~120°. The
holder is tilted incrementally around an axis that is
perpendicular to the electron beam. All tilted projections are
synthesized into a 3D density map, typically by applying a
‘weighted back-projection’ algorithm (Radermacher, 1992).
Shown are projections and a reconstructed volume of a
D. discoideum cell, adapted from Medalia et al. (Medalia et
al., 2002). Scale bar: 300 nm. (B)Surface-rendering view of
the reconstructed volume shown in A shows the actin
filaments (red), cell membrane (purple) and large
macromolecular complexes, mostly ribosomes (green). The
surface-rendering views were segmented semi-automatically.
Colors were chosen subjectively. Branches of actin filaments
as found at the cell cortex are shown in the lower panel.
(C)Surface-rendering view of the reconstructed volume of a
human fibroblast cell shows all cytoskeletal elements. Cortical
actin (red) is located along the cell membrane (purple),
whereas intermediate filaments (turquoise) are localized
further into the cell interior; these present a wider diameter
(~10 nm), a different texture and a lower persistence length
than actin. In addition, one microtubule (pink) is found in the
upper left corner of the tomogram in close proximity to a
cluster of ribosomes (green). Scale bar: 100 nm (also for B).
(B)Adapted from Medalia et al. (Medalia et al., 2002).
Journal of Cell Science
Cryo-ET of eukaryotic cells
in highly motile cells, the proteins associated with cytoskeletal
networks have half-lives in the order of 7-10 seconds (Bretschneider
et al., 2004). Therefore, it is to be expected that actin-bundling and
-crosslinking proteins, which are crucial for maintaining the
architecture of the actin network, might dissociate from actin
filaments and redistribute within a specimen during the course of
traditional-preparation EM procedures.
In live cells, actin structures rapidly reorganize during motility,
endocytosis and cytokinesis (Dalous et al., 2008; Kaksonen et al.,
2004; Pantaloni et al., 2001; Pellegrin and Mellor, 2007; Walpita
and Hay, 2002). Because of these dynamics, immediate arrest of
the cellular processes in intact cells is a prerequisite for obtaining
faithful information regarding actin networks. Electron tomography
of vitrified but otherwise unaltered cells has proven to be a key
technique for the 3D reconstruction of actin architecture (Medalia
et al., 2002). It was demonstrated that cryo-ET of intact
Dictyostelium discoideum cells could reveal the connections of the
actin-filament network with the plasma membrane (Fig. 1B, upper
panel), as well as massive actin filaments branching at various angles
(Fig. 1B, lower panels), without the need for chemical fixation or
heavy-metal decoration (Medalia et al., 2002). Similar views can
be obtained when cells of higher eukaryotes are studied (Fig. 1C).
In the future, our understanding of cell motility and other
cytoskeleton-dependent processes will be increased by investigating
the 3D organization of the actin system in relation to specific
functional states, such as during consecutive steps of particle uptake
by a phagocyte or at cell-adhesion sites. The possibility of arresting
cells instantly also allows the investigation of rapid cellular
processes such as filopodial protrusion, which is discussed below.
Zooming in on actin remodeling during
filopodium formation
Filopodia are finger-like plasma-membrane protrusions that are
involved in adhesion to the extracellular matrix (ECM), sensing the
environment and cell-cell signaling (Chhabra and Higgs, 2007; Mattila
and Lappalainen, 2008). Filopodia also represent an excellent model
system for describing the process of actin-driven membrane
protrusion. These structures grow at their tips through the assembly
of actin and are stabilized along their lengths by a core of bundled
actin filaments (Pollard and Borisy, 2003). Given their relatively low
thickness (150-400 nm), filopodia are also excellent cellular structures
for study by cryo-ET. With this approach, the organization of actin
filaments and membranes to which the actin network is anchored can
be carefully analyzed in an unperturbed state.
We used cryo-ET to track the length and relative position of
individual filaments within D. discoideum filopodia, which allowed
a quantitative analysis of actin filaments. The data revealed that
actin filaments in these fast-moving cells are not continuous
throughout the entire protrusion (Fig. 2) (Medalia et al., 2007).
Importantly, it was shown that the filopodial tip comprises many
short filaments that interact with the membrane at their distal and
proximal ends. These filaments, arranged at the tip in a cone shape,
have been suggested to provide the driving force that pushes the
membrane forward (Gerisch and Weber, 2007). Their location and
length support the notion that sites of de novo actin-filament
nucleation and growth are confined to the tip of the filopodia (Faix
et al., 1992; Faix and Grosse, 2006; Medalia et al., 2007). It is
noteworthy that no vesicles are found within filopodia that are
visualized by cryo-ET, implying that the membranes needed for
the formation of protrusions are supplied at a distance from the
filopodial tip. The presence of Dia2, a formin with actin-nucleating
9
Fig. 2. The architecture of D. discoideum filopodia. (A)A 50-nm
tomographic slice through a filopodium demonstrates the discontinuity of the
filopodial actin filaments. Short filaments are found at the tip of the
filopodium, and these are distinct from the transverse and straight filaments
found along the filopodial shaft. Scale bar: 200 nm. (B)Surface-rendering
view of the filopodium (boxed area in A) reveals the overall organization of
the actin network (red) and the interaction of actin filaments with the plasma
membrane (blue) at the filopodial tip. Macromolecular complexes are shown
in green. (C)The ‘sequential-nucleation model’ (see text) is illustrated. At the
tip of the filopodium, de novo nucleation and growth of actin filaments occur
(Medalia et al., 2007). In the shaft zone, actin filaments are bundled and
axially oriented along the filopodium and, within the tip-shaft zone, growing
actin transverse filaments are connected to the membrane and to the shaft
filaments. Adapted from Medalia et al. (Medalia et al., 2007).
activity that is important for filopodia formation and maintenance
(Schirenbeck et al., 2005), at the tips of filopodia in D. discoideum
implies that actin filaments undergo nucleation through filopodia
protrusion. When filopodia protrude, the actin filaments grow and
are then bundled while laterally connecting to the membrane along
the filopodial shaft (Medalia et al., 2007). In general, we found that
D. discoideum filopodia are characterized by a discontinuity of actin
filaments along the filopodial axis (Fig. 2C). However, some
transverse filaments connect to the membrane and the shaft filaments
are found in the tip-shaft zone.
This analysis can thus be explained by the ‘sequential-nucleation
model’ (Medalia et al., 2007), which proposed that the sites of de
novo nucleation and growth of actin filaments are confined to the
filopodium tip. The short filaments located at the tip detach from
and reattach to the cell membrane with their distal and/or proximal
ends, thus enabling actin polymerization. The growing filaments
are then bundled and laterally connect to the cell membrane along
the filopodia shaft. Within the shaft zone, actin filaments are bundled
and axially oriented (Fig. 2C). The unique organization and
arrangement of D. discoideum filopodia can presumably be
attributed to the fast motility of these cells. That is, the apparent
discontinuity of actin filaments might be a property of filopodia in
10
Journal of Cell Science 123 (1)
Journal of Cell Science
Fig. 3. Surface-rendering view of a Ptk2 cell infected by HSV-1. Two capsids
of recently entered virions (light blue) were found to reorganize and modify the
actin bundles, but not depolymerize actin, upon viral entry. On the upper left
side, the virus-derived glycoprotein spikes (yellow) can be seen emerging from
the membrane. Viral tegument (orange), cell and viral membrane (dark blue),
actin (dark red; upper part cut away), and cellular vesicles (purple) are shown.
Adapted from Maurer et al. (Maurer et al., 2008). Scale bar: 100 nm.
fast motile cells, which differs from filopodia in other adherent cells,
which are characterized by continuous actin filaments.
Remodeling of actin networks during viral entry
Several viruses use an endocytic mechanism to enter cells prior to
remodeling cortical actin (Greber, 2002; Munter et al., 2006). In
addition, filopodia and other cellular protrusions are susceptible to
viral docking, which eventually leads to cell infection (Clement et al.,
2006). Cryo-ET can provide a unique tool for studying infected cells,
supplying unprecedented information on the stages of viral assembly
and maturation within cells. In a pioneering study, Maurer et al. showed
snapshots of the entry of Herpes simplex virus 1 (HSV-1) into cells
(Maurer et al., 2008). By means of cryo-ET, the authors showed that
cytosolic capsids of HSV-1 were located between actin bundles within
PtK2 cells. They also identified capsids between individual actin
filaments in the cell cortex. Furthermore, the data revealed that the
virus does not induce local depolymerization of actin in its vicinity,
on the basis of morphological appearance of the cell cortex, but rather
might be involved in remodeling the dense cytoskeletal network as
shown by Clement et al. (Clement et al., 2006) (Fig. 3).
Fig. 4. Cryo-ET of the nuclear envelope. (A)A 32-nm tomographic slice
through the nuclear envelope of a human fibroblast shows a central slice through
an NPC, which fuses the inner and outer nuclear membranes (INM and ONM,
respectively). Scale bar: 100 nm. (B)Stereo-view representation of an averaged
reconstructed volume of the human NPC. The central spoke ring is flanked by
the cytoplasmic ring (arrowheads) and the nuclear ring. (C)Schematic
representation of the nuclear envelope on the basis of cryo-ET of intact nuclei.
The ONM is decorated with ribosomes (red), and the ONM and INM (yellow)
are fused at the NPC (blue). Nuclear lamins (purple) are seen underlying the
INM and interacting with the NPC via the nuclear basket (green). The structure
of the lamin filaments was adapted from an in vitro cryo-ET study of the
Caenorhabditis elegans lamin filaments (Ben-Harush et al., 2009).
The NPC: combining cryo-ET and singleparticle approaches
Cryo-ET is primarily a static tool. However, by collecting a large
number of datasets and correlating them with pre-existing
information, one can acquire information about a dynamic process
at the molecular level, as has been described above for filopodia
and virus-infected cells. Thus, the remodeling and structural changes
of macromolecular complexes that transpire during cellular
processes can be examined. Additionally, a detailed 3D
reconstruction of macromolecular complexes in situ can be achieved
by combining cryo-ET with 3D-averaging procedures (Bartesaghi
et al., 2008; Bostina et al., 2007; Forster et al., 2005).
Such a hybrid technique (cryo-ET and 3D-averaging approach)
was applied to NPCs, which are large molecular machines that are
embedded in the nuclear envelope and connect the nucleoplasm with
the cytoplasm by means of an aqueous channel. NPCs, which are
composed of hundreds of proteins (Alber et al., 2007) arranged in
pseudo-eightfold rotational symmetry, function as a selective barrier
(Rout et al., 2000) by allowing small molecules and ions to diffuse
freely while mediating the passage of large molecules in an energydependent manner. Although work on the NPC structure using EM
began in 1950 (Callan and Tomlin, 1950), only at the end of the
twentieth century were the main components of the structure revealed
(Akey and Radermacher, 1993; Brohawn et al., 2009; Yang et al.,
1998). Despite dimensional differences between species, the basic
architecture of the NPC is conserved; the consensus structure consists
of a central spoke ring that is confined by a cytoplasmic and a
nucleoplasmic ring. Eight cytoplasmic filaments and a nuclear basket
composed of eight filamentous structures join to form a distal ring.
Owing to its sheer size, the NPC presents a major challenge for
structural determination. Applying cryo-ET to intact nuclei ensures
that the NPC is arrested in its active form, as is evident from the
preservation of a nucleocytoplasmic gradient of Ran-GTP in isolated
nuclei (Becskei and Mattaj, 2003; Becskei and Mattaj, 2005; Gorlich
et al., 2003). The nuclear envelope can be viewed as an ellipsoid
Journal of Cell Science
Cryo-ET of eukaryotic cells
with NPCs embedded in its surface in all possible orientations; thus,
extracting these elements in silico, followed by 3D alignment and
averaging, results in isotropic resolution, i.e. in all three dimensions.
Using cryo-ET, we have resolved the NPC to 8-9 nm, in which some
of the flexible filaments of the NPC were observed, in addition to
the scaffolding features of the complex (Beck et al., 2004). An
improvement in resolution (<6 nm) was later achieved when the
structure of the D. discoideum NPC was resolved without imposing
eightfold symmetry (Beck et al., 2007). In this study, snapshots of
the trajectory of cargo transported through the NPC were visualized.
Recently, work from our laboratory has shown that the application
of a similar approach using nuclei from human fibroblasts (Fig. 4)
yielded the first insight into the structure of the human NPC (Elad
et al., 2009). Although the resolved structures of the D. discoideum
NPC and the preliminary structure of the human NPC share several
common features, such as the outer and inner diameters (~120 nm
and ~50 nm, respectively), they differ in height and protein-density
distribution, suggesting differences in protein positions (Elad et al.,
2009). Currently, analysis of intact nuclear envelopes by means of
cryo-ET is limited to a resolution of 5-8 nm, which restricts structural
interpretation to the level of determining the position of subcomplexes.
Increasing the resolution of tomograms and the application of
specimen-thinning techniques, such as cryosectioning (see above) and
focused ion beam (see below), will enable a step forward in
understanding the functional organization of the nuclear envelope.
Conclusions and perspectives
Cryo-ET is the method of choice in acquiring an insight into the
molecular organization of cells and cellular components, such as
filopodia and actin filaments at the cortex of the cell, and to track
the entry of viruses into cells. Additionally, it allows determination
of the 3D structure of large supramolecular assemblies in situ, as
we demonstrated for the NPC, at a medium resolution of 4-6 nm.
A major challenge facing cryo-ET concerns the identification of
macromolecular complexes within a cellular context. The different
orientations of macromolecules and the current resolution of cellular
tomograms prohibits unbiased identification of many molecular
complexes, although some successful template-matching approaches
have been introduced (Frangakis et al., 2002; Ortiz et al., 2006).
These procedures aim to identify specific macromolecular
complexes in vivo on the basis of their structural fingerprint, by
searching for in-vitro-determined structural complexes in a
tomogram (van Heel et al., 2000). An approach based on electrondense labeling must be developed to design a clonable tag that can
be genetically conjugated to proteins, which would facilitate their
localization in cryo-tomograms – that is, we are in need of a GFP
analog for cryo-electron microscopy. An elegant example of a
clonable tag is metallothionein, a cysteine-rich protein that has been
shown to bind to multiple heavy atoms and can be detected by an
electron beam (Mercogliano and DeRosier, 2007). Such a labeling
strategy would provide a general solution for identifying complexes
whose structure is not yet determined or that intimately interact
to form large assemblies. To make cryo-ET applicable not only to
cellular protrusions and thin regions of the eukaryotic cell but also
to thicker samples, there is a need to develop a reliable freezehydrated artifact-free sectioning technique that can be applied to
tissues and cells to produce optimal (thinner than 500 nm) biological
samples for cryo-ET. Alternative micro-dissection techniques that
involve using a focused ion beam to mill frozen samples are
currently being developed. In these approaches, gallium ions (Ga+)
are directed onto the frozen cell or tissue sample at a specific angle
11
and, by process of ‘sputtering’, selected parts of the sample can be
removed; this is known as ion-beam milling. Notably, this process
does not cause major artifacts below 30 nm from the upper level
of the milled surface (Marko et al., 2006; Marko et al., 2007).
Realization of this technology would open a window for the entry
of cryo-ET into other branches of biology and might provide, for
instance, a bridge between structural and developmental biology.
Another direction of technical development lies in correlating
fluorescent and cryo-electron-microscopy images; this would greatly
help in identifying attractive locations within the cell for
investigation by cryo-ET (Sartori et al., 2007; Schwartz et al., 2007),
and would allow for the reconstruction of important cellular
structures. This approach would permit the identification of specific
states of cellular processes and would therefore eventually produce
structural snapshots of such processes. Moreover, developing
automated fast algorithms would allow for a shortening of current
time-consuming procedures, making analysis more robust.
In the future, the application of other correlative approaches – such
as combining cryo-ET with atomic force microscopy – would also
enable correlations between physical changes in the cell, force
measurements and structural information. It is expected that such
hybrid methods will lead to platforms that can provide deeper insight
into cellular processes. Complementary information from a variety
of techniques will thus be combined to reconstruct meaningful cellular
density maps (Robinson et al., 2007). With advanced instrumentation,
such as advanced charge-coupled device (CCD)-camera detectors and
dual-axis tilting devices, the prospects are good that higher and more
isotropic resolutions of 2-3 nm can be attained. Therefore, we foresee
that cell biology will increasingly rely on high-resolution 3D imaging
techniques, in conjunction with other approaches.
This work was supported by a grant from the German-Israeli
Cooperation Project (DIP) (H.2.2), by the Israel Science Foundation
(grant 794/06) and by the German-Israel Foundation, to O.M. We thank
Kay Grünewald and Ulrike Maurer for providing Fig. 3.
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