REVIEW ARTICLE

Molecular Reproduction & Development 82:530–547 (2015)

Three Dimensional Reconstruction by Electron Microscopy in

the Life Sciences: An introduction for Cell and
Tissue Biologists
KILDARE MIRANDA,1,2* WENDELL GIRARD-DIAS,1 MARCIA ATTIAS,1 WANDERLEY DE SOUZA1,2,
3
AND ISABELA RAMOS *

1

, rio de Ultraestrutura Celular Hertha Meyer, Instituto de , Biof
ısica Carlos Chagas
Laborat, o
^, ncia e Tecnologia em Biologia Estrutural e Bioimagens
Filho and Instituto Nacional de Ci, e



Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
2
Diretoria de Metrologia Aplicada a Cie ^ncias da Vida, Instituto Nacional de Metrologia,
Qualidade e Tecnologia (INMETRO), Xer, e
, m, Rio de Janeiro, Brazil
3
Laboratorio de Bioquımica de Insetos, Instituto de Bioqu
ımica Me



dica Leopoldo de Meis



Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

SUMMARY
Early applications of transmission electron microscopy (TEM) in the life sciences
have contributed tremendously to our current understanding at the subcellular level.
Initially limited to two-dimensional representations of three-dimensional (3D) objects,
this approach has revolutionized the fields of cellular and structural biology
being A number of techniques are
instrumental for determining the fine morpho-functional characterization of most available to generate volumetric
cellular structures. Electron microscopy has progressively evolved towards the ultrastructural models, and
development of tools that allow for the 3D characterization of different structures. combination of a variety of
This was done with the aid of a wide variety of techniques, which have become strategies is now possible for
increasingly diverse and highly sophisticated. We start this review by examining the
tailoring to specific biological
principles of 3D reconstruction of cells and tissues using classical approaches in
TEM, and follow with a discussion of the modern approaches utilizing TEM as well as questions and applications.
on new scanning electron microscopy-based techniques. 3D reconstruction techni-
ques from serial sections and (cryo) electron-tomography are examined, and the 
Corresponding authors: Laboratorio de
recent applications of focused ion beam-scanning microscopes and serial-block-face Ultraestrutura Celular Hertha Meyer
Instituto de Biofısica Carlos Chagas
techniques for the 3D reconstruction of large volumes are discussed. Alternative low- Filho and Instituto Nacional de
cost techniques and more accessible approaches using basic transmission or field Ci^encia e Tecnologia em Biologia
emission scanning electron microscopes are also examined. Estrutural e Bioimagens Universidade
Federal do Rio de Janeiro Rio de
Janeiro, Brazil. E-mail:
kmiranda@biof.ufrj.br (K.M.);
isabela@bioqmed.ufrj.br (I.R.)
Mol. Reprod. Dev. 82: 530
547, 2015. ß 2015 Wiley Periodicals, Inc. Published online 4 February 2015 in Wiley Online Library
(wileyonlinelibrary.com).
Received 1 July 2014; Accepted 10 December 2014 DOI 10.1002/mrd.22455

INTRODUCTION
Understanding the functional organization of cells and
tissues is a hallmark for understanding life. The description
of cell and tissue organization and the discovery of novel
Abbreviations: 2/3D, two/three dimensional; ART, algebraic reconstruction
techniques; ATUM, automated tape-collection ultramicrotomy; CCD, charge-
coupled device; FIB, focused ion beam; SBF, serial block face; SEM, scanning
electron microscope; SIRT, simultaneous iterative reconstruction technique;
TEM, transmission electron microscope; WBP, weighted back-projection

ß 2015 WILEY PERIODICALS, INC.

 THREE DIMENSIONAL RECONSTRUCTION BY ELECTRON MICROSCOPY
structures at the micro- and nanoscale occurred in parallel
with the development of several microscopy techniques.
Most of these techniques initially provided information on
the two-dimensional (2D) organization of the object. Devel-
opment of techniques that added a third dimension, how-
ever, allowed for a more complete assessment of biological
structure and revolutionized the fields of cell and tissue
biology. Like light microscopy, electron microscopy has
taken a similar path of maturation, and continues to evolve
through technology that provides subcellular access to the
third and fourth dimensions (Lorenz and Zewail, 2014).
Advances in three-dimensional (3D) electron microsco-
py have attracted those biologists who want to explore the
volumetric architecture of their models at high resolution. A
variety of new approaches are available, yet all options are
grounded with basic knowledge generated in the early days
of electron microscopy, which provided the foundation for
the development of what is today considered cutting-edge
methodology. Of course, the question of which method is
best suited to a model emerges from the array of possibili-
ties. The answer is sometimes not straightforward, and a
combination of methods may eventually be chosen to
address a single question.
The introduction of the electron microscope as a re-
search tool in biology, therefore, has provided a complete
reassessment of the microanatomy of biological tissues,
its cells, organelles, and molecules. One of the main
challanges faced by the life-sciences research field in
the early days of electron microscopy was the sensitivity
of biological samples to the electron beam, which usually
operates at high-acceleration voltages and high energies.
Enormous improvements for biological electron micros-
copy came with advances in sample-preparation methods
that provided stability and specific contrast to different
samples according to the type of study required (e.g.,
routine methods, enzyme cytochemistry, immunogold
labeling, and autoradiography). Different sample-prepa-
ration techniques were then developed, thus establishing
a new research field that continues to evolve (Bozzolla
and Russel, 1992). Before going into detail on the sample
preparation and advances in analysis techniques, we
must first briefly discuss the fundamentals of single-plane
transmission electron microscopy, which is performed
using the most basic instruments.
BASIC PRINCIPLES OF TEM IMAGING
The transmission electron microscope (TEM) is a pro-
jection instrument. It operates using an accelerated elec-
tron beam that passes through a series of magnetic lenses
(condensers, objectives, and projectors), interacts with the
sample, and projects an image to a reactive surface (e.g., a
fluorescence screen, film, or digital camera). The image
formed is the result of an interaction between the electron
beam and a sample (usually a thin section). A number of
phenomena are involved in this interaction

in particular,
the electrons can be differentially scattered in a predictable
manner that depends on the structure of the sample (re-
Mol. Reprod. Dev. 82:530–547 (2015)
viewed in Bozzolla and Russel, 1992). From a reductionist
perspective, regions of the sample that contain heavy
elements (high atomic number) or have higher mass den-
sity will tend to scatter the incident electrons more, which
will then be retained or blocked by microscope components
(apertures), resulting in darker regions on the projected
image.In the case of biological samples, this is achieved by
using a number of staining agents during sample prepara-
tion (osmium tetroxide, uranile acetate, lead citrate, e.g.). In
addition, except for some special techniques where high
voltage and/or energy-filtered TEMs are used to image
whole cells (Miranda et al., 2000, 2010), for most applica-
tions, ultrathin (below 100
nm) samples have to be used
(Bozzolla and Russel, 1992). In the case of room tempera-
ture analysis of suspensions of particles, filaments, isolated
organelles and small cells (proteins, viruses, cytoskeleton
elements, etc), negative staining is usually the method of
choice (Brenner and Horne, 1959). For most cell types,
ultrathin sectioning (Porter and Blum, 1953) of resin em-
bedded samples is used (Glauert et al., 1956; Maale and
Birch-Andersen, 1956). The implications of sectioning the
samples and how it limits the amount of information that can
be obtained from an originally 3D object will be discussed in
the next sections.
TRANSMISSION ELECTRON MICROSCOPY-BASED
APPROACHES FOR 3D RECONSTRUCTION
The Classic Approach: 3D Reconstruction From
Serial Sections
Obtaining volumetric information by electron microsco-
py has been a challenge since the first demonstrated
biological applications of this technique. The TEM has
been the primary tool used for the ultrastructural analysis
of different cell types. A combination of techniques has
been developed that can provide additional information,
including 3D reconstruction (Crowther et al., 1970; Hoppe
and Grill, 1977; Frank, 2006). Examination of most cell
types by a TEM usually requires a number of sample
preparation steps that involve chemical fixation, embed-
ding, and ultra-thin sectioning. Low penetration of the
electron beam and aberrations (that increase with sample
thickness) limits samples to a thickness of 60
100 nm,
which severely restricts the sampling volume. Planar (2D)
images of 3D structures that can extend for several micro-
meters in all directions are obtained, thus presenting a
small fraction of the whole sample volume while neglecting
information on the 3D structural organization of the speci-
men (Frank, 2006; Chklovskii et al., 2010).
A number of techniques to circumvent the limitation of
image acquisition are available (summarized in Fig. 1), with
some requiring the use of sophisticated equipment such as
medium- and high-voltage microscopes or dual-beam
scanning electron microscopes (SEMs). Such specialized
equipment is not yet implemented in most laboratories or
microscopy centers, so the application of more-traditional
approaches using a regular TEM has become one solution
that can result in valuable 3D information when combined
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Molecular Reproduction & Development MIRANDA ET AL.

Figure 1. Scheme showing different approaches to 3D electron microscopy image acquisition. A: Serial-section transmission electron microscopy.
B: Electron tomography. C: Serial-section scanning electron microscopy. D: SBF scanning electron microscopy. E: Serial FIB-milling. In all cases,
the steps of 3D image acquisition involve the acquisition of a misaligned image series (tilt-series in the case of electron tomography); image stacking
(3D reconstruction); and alignment of the series, segmentation, and modeling. In electron tomography, it is necessary to perform the reconstruction
of the tilt series after alignment, which can be achieved by different approaches such as WBP, SIRT, and ART. SEM methods (C
E) are grouped by
their final resolution (intermediate), where serial-section scanning electron microscopy (C) and SBF scanning electron microscopy (D) have roughly
the same zeta resolution, whereas serial FIB-milling (E) produces slightly better zeta resolution.

532 Mol. Reprod. Dev. 82:530–547 (2015)

 THREE DIMENSIONAL RECONSTRUCTION BY ELECTRON MICROSCOPY
with methods, such as serial sectioning (Williams and
Kallman, 1955; Ward et al., 1975; Coombs et al., 1986;
Attias et al., 1996; Shoop et al., 2002; Miranda et al., 2004).
3D reconstruction from serial sections is defined by the
acquisition and reassembly of images from different pro-
files of the same cell in sequential sections (Figs 1 and 2). A
sample structure can be followed along the depth of the
stacked sections, and the subsequent application of image
processing, reconstruction, alignment segmentation, and
modeling steps help generate a 3D model (Figs. 3 and 4).
Volumetric, 3D reconstruction from serial sections using a
TEM is considered a low-resolution technique since the
projection of structures distributed along the sections depth
to a 2D plane results in a fuzzy image that contains mixed
information of overlapping structures that can corrupt the
quality of the individual images in a series. The zeta reso-
lution (resolution in the z axis) of a model is, therefore,
limited to the section thickness (McEwen and Marko, 1999).
TEM analysis of serial sections also requires the acquisition
of ribbons of consecutive sections that are obtained using
an ultramicrotome and collected on specially coated elec-
tron microscope grids that contain a central slot (Fig. 2). The
lenght of this slot limits the total volume that can be ana-
lyzed to the sum of the thicknesses of each section con-
tained in the grid (typically 30), unless consecutive ribbons
are collected in different grids (see below) (White et al.,
1986; Bumbarger et al., 2013). For these reasons, TEM-
based 3D reconstruction from serial sections is generally
applied to what may be considered intermediate volumes
when compared to the other techniques discussed in this
review (see below).
The shape of the block surface (face) used in routine
ultramicrotomy is not ideal for the serial sectioning required
in 3D electron microscpy. In normal ultra-thin sectioning for
Figure 2. Example of a serial TEM acquisition. A: Slot grid containing a ribbon of serial sections. B
J: A sequence of images acquired from serial
sections of differentiating monocytes. Scale bars, 0.5 mm (A) and 5 mm (B
J). Adapted with permission from Miranda et al., 2011.
Mol. Reprod. Dev. 82:530–547 (2015)
2D analysis, the block narrows towards the plane parallel to
where the sectioning knife enters the block, resulting in the
rapid increase in cross-sectional area within a few conse-
cutive ribbon sections; this increased area could ultimately
cover the whole extension of the grid, thereby restricting
number of sections per ribbon per grid and severely ham-
pering the analyzable volume (Fahrenbach, 1984). To
overcome this limitation, a wider face with a shorter height
than usual, with the side faces and the basal angles less
than 608 (508
408), should be sculpted from the sample
block, which allows more sections to be placed in one grid
since the section area would slowly increase with the
progression of ultramicrotomy (Fahrenbach, 1984). An
alternative method for preparing the block face using a
glass knife and an ultramicrotome can be used, resulting in
up to 100 sections in a slit of 2 mm in length (Elliot, 2007). A
few groups have gone through tremendous effort to collect
an impressive number of consecutive sections (up to 3,000)
in different slot grids, a process that is generally time-
consuming (White et al., 1986; Bumbarger et al., 2013).
Once a ribbon of sections is obtained, simple and careful
observation of profiles of the same structure in serial
sections may, in many cases, lead to the understanding
of its 3D structure. When querying long series of sections
for complex structures, however, it is not easy to envision
the 3D volume by simply observing micrographs; instead, it
becomes mandatory to generate a 3D model that can be
rotated in all directions so that a correct volumetric repre-
sentation can be obtained. In early work, such models were
generated by sculpting the structures found in each micro-
graph using a variety of materials such as clay, Styrofoam,
acrylic, or any transparent material, followed by their as-
sembly into a physical, 3D model (Gaunt, 1978; Fisher
et al., 1979; Stephens, 1979; Kinnamon and Westfal,
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Molecular Reproduction & Development
Figure 3. Manual alignment of serial images. A: The merged overlap of two serial images with different color codes (green and pink), which shows
misaligned edges. B: Merged outcome of two aligned images, where the sum of the color codes provides a less-colored image. Scale bar, 50 mm.
1981). Although simple by design, implementation of these
methods was extremely laborious and time-consuming,
and the models were often limited. Nevertheless, important
information was obtained using this approach, such as the
concept of the single mitochondrion in some cell types
(Paulin, 1977), chloroplasts and mitochondria in Eugle-
noids (Pellegrini, 1980), and the mitotic apparatus of Try-
panosomatids (Solari, 1983).
With the introduction of digital imaging (initially using
scanned film negatives, followed by image acquisition with
a charge-coupled device [CCD] camera), software for 3D
reconstruction and modeling was developed such that
volumetric modeling from digital images obtained from
serial sections became an accessible technique for any
electron-microscopy laboratory. Some of the best recon-
struction software currently available is free (see below).
3D reconstruction and generation of digital models follows a
routine pipeline of image acquisition and processing steps
(summarized in Fig. 1) that comprise: (1) selection of the
structure of interest in different sections of the series; (2)
image acquisition of the structure of interest in the whole
series; (3) alignment of the images; (4) segmentation; and
(5) generation of models. The resulting model can then be
rotated in different directions, structures can be shown or
hidden for a better presentation of the results, and movies
showing different angles of the structures of interest can be
generated.
As each image is obtained from sections that are physi-
cally separated, small angle differences as well as small
deformations (namely shrinking) due to beam damage
(bombardment with the electron beam) frequently lead to
misalignments that can be corrected or minimized with the
use of specific alignment tools available in the software.
Some of these alignment tools may be applied automati-
cally (e.g., cross-correlation) or manually by comparing two
sequential images in a series (Fig. 3). Once an aligned
sequence of images is obtained, a segmentation step that
generates contours from the image is required to assign a
pixel identity to the profiles of the structures of interest in
each section. This step consists of outlining structures of
the sample by manually segmenting structures of interest in
each 2D image that contributes to the volume. Contours
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MIRANDA ET AL.
representing the same type of structure (plasma mem-
brane, nuclear envelope, and etcetera) are then intercon-
nected in a process of volume rendering that gives birth to
the 3D model (Fig. 4). When the contrast of the structures of
interest is sufficiently different from all other structures in
the volume, segmentation can be done automatically by
assigning intensity values to groups of pixels according to
certain threshold limits defined by the user (see below).
Such automatic segmentation is the fastest method of
generating 3D models, although they often do not work
well in reconstructions of biological samples due to the
heterogeneous distribution of intensities in the sample.
Electron Tomography
As mentioned above, 3D reconstruction from serial
sections remains an essential tool for many laboratories,
despite its limitations when compared to other 3D recon-
struction approaches. Some of these drawbacks include
limited sample volume due to the maximum number of
sections that can be collected in a grid, thickness variations
during the ultramicrotomy process, distortions caused by
the uneven heating of sections by the electron beam, and
low zeta resolution due to the thickness of the sections
(Frank, 1995). The development of medium- (200
400 kV)
and high-voltage (1000
3000 kV) electron microscopes
allowed for the observation of specimens up to 50 times
thicker (0.2
2 mm) than the sections observed in con-
ventional TEMs (Wilson et al., 1992; Martone et al., 2000).
Although remarkably rich in the number and diversity of
structures that can be observed in the thicker sections, the
resulting images are difficult to interpret since the frequency
of structure overlap is much greater, causing the single,
projected image to be fuzzier (Bouwer et al., 2004). Visual-
ization of 3D structures of large volumes in high-voltage
TEMs was nonetheless carried out with the use of stereo-
pairs, a combination of two images of the same object taken
at small angle differences (usually 58
158) (Ris, 1981;
Clermont et al., 1995).
High-resolution 3D analysis of cells and subcellular
structures remained challenging until the development of
electron tomography (Klug and De Rosier, 1966; De Rosier
Mol. Reprod. Dev. 82:530–547 (2015)
 THREE DIMENSIONAL RECONSTRUCTION BY ELECTRON MICROSCOPY
Figure 4. Steps required to generate a 3D model from serial images acquired by serial-section transmission electron microscopy of a differentiating
monocyte. A: TEM image of a cell. B and C: Manual segmentation of the nucleus in different sections. D
F: 3D model of the nucleus (N) shown in
panel A. Scale bar, 5 mm.
and Klug, 1968), a highly sophisticated approach that some
may consider an ellaborated extension of the stereopair
approach employed in high-voltage TEM analysis. Electron
tomography consists of acquiring a tilt series of projected
images in the electron microscope (see below), followed by
a number of image processing and digital reconstruction
steps that generate a 3D volume. As the volume generated
is composed of consecutive digital sections, it is possible to
extract high-resolution information from individual planes
(e.g., 1-nm digital sections) (Koster et al., 1997; Lucic et al.,
2005; Frank, 2006; Barcena and Koster, 2009). The volu-
metric model can then be virtually sectioned, wherein the
information above and below the sample is excluded to
exclusively view the selected plane, resulting in very sharp
images of the structures without the inference of structures
contained in adjacent image planes. For this reason, elec-
tron tomography is considered a high-resolution 3D recon-
struction technique.
The principles of electron tomography are similar to
those applied in computed tomography for medical diag-
nostics. Essentially a 3D body composed of regions of
variable density is traversed by a radiation (X-rays, light,
or electrons) tilted at different angles, from which the
intensity before and after hitting the sample at these com-
parative angles can be measured. Differences between
Mol. Reprod. Dev. 82:530–547 (2015)
intensities result from absorption of the radiation by the
material within the sample. The theoretical principles of
tomography were established by Radon (1887
1956), a
German mathematician who developed the ‘‘Radon trans-
form’’. Haunsfield Cormack applied Radons concepts to
develop computed tomography (or ‘‘CT’’), for which he was
awarded the Nobel Prize for Medicine in 1979. Since it is
possible to accurately calculate the relative intensity differ-
ence of the radiation that passes through an object, one can
reconstruct the interior of a sample by irradiating it at
different angles and collecting data from each angle. The
mathematical concept proposed for tomography is the
reconstruction of a 3D volume from 2D projections of an
object at various angles.
In electron tomography, semi-thin sections (0.2
2 mm)
of a selected sample are used. Once the cell or region of
interest is selected, the specimen is rotated around its y-
axis, at defined incremental angles through an angular
range (typically
708
þ708). Images are acquired, usu-
ally via CCD cameras connected directly to the micro-
scope, where the series will be recorded for later
tomographic alignment and 3D reconstruction in specific
software. Semi-thin sections, thicker than those used in
regular 2D TEM, are used so that a larger volume of the
sample can be analyzed and reconstructed, although the
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Molecular Reproduction & Development
voltage of the electron microscope ultimately dictates the
maximum section thickness that can be used. For in-
stance, for section thicknesses of 200
500 nm up to
1 mm, it is possible to use scanning TEMs (see below)
with an intermediate voltage (200
300 kV). This is due to
the fact that as the tilt angle of the sample is increased
during the tomogram acquisition, its thickness increases:
a 200-nm thick sample at 708 tilt angle becomes 2.924
times thicker (585 nm). As higher voltages elicit better
penetration of the electron beam in the sample, using
more powerful TEMs will reduce some optical aberrations
associated with excess inelastic interactions that come
from the thicker specimens, thus providing an image with
less chromatic aberration and, consequently, higher res-
olution and contrast. On the other hand, as the thickness
increases, a sample will suffer more damage by
irradiation.
Special specimen holders with a conical shape have to
be used for electron tomography since the use of regular
holders may block the electron beam when the specimen is
tilted at high angles ( 708). Likewise, if the spacing be-
tween the grid bars is too small (<300 mesh), the tilt angle
will also be reduced because the grid bar will also block the
electron beam (before the tilting limit is reached by the
holder). In this case, it is preferable to use grids of 200 mesh
or less. A regular tilt series employs a tilt angle limited to
 708. The information lost from higher tilt angles, however,
introduces a problem called the ‘‘missing wedge’’; since
resolution is improved with larger numbers of angles, this
missing wedge handicaps zeta resolution of the recon-
structed tomograms (Frank, 2006). This problem can be
reduced by acquiring two tilt series from the same sample
area by manually turning the grid 908, by using a sample
holder that allows the rotation of the grid, or by employing
robotics; this orthogonal acquisition approach is called
dual-axis tomography (Mastronarde, 1997). Better cover-
age of the missed information can be achieved with other
acquisition strategies, such as conical tilting, which ac-
quires the different tilt series at several tilt axes by auto-
matic rotation of the grid (Radermacher et al., 1987;
Lanzavecchia et al., 2005; Zampighi et al., 2005).
Electron tomography experienced a great improve-
ment in recent years, mainly due to the increase in
processing power of computers and advances in compu-
tational techniques for automatic control of the acquisi-
tion of tomographic series (Typke et al., 1991; Koster
et al., 1992; Koster et al., 1992; Koster and de Ruijter,
1992; Typke et al., 1992). The latter refers to the substi-
tution of operations normally performed by the user in
the microscope by a fully automated method, with little or
no interference of the user after starting an acquisition.
Once the sample is inserted into the TEM, a series of
images at different tilt angles is acquired. As the sample
is tilted, small shifts and focus changes due to mechani-
cal imperfections of the goniometer and the holder will
occur. These changes may be corrected or minimized
with the use of automated functions available in the
software, although a more accurate alignment of 2D
images should be performed after the series acquisition
536
MIRANDA ET AL.
is complete (post-acquisition alignment). This correction
procedure is carried out using a sequence of digital filters
to correct frequencies, cross-correlation functions and
Fast Fourier Transforms to correct for sample shifts,
changes in sample height, focus displacements, and
contrast transfer function correction (for details, see
Frank, 2006).
Post-acquisition alignment can be done in different
ways. The strategies practiced by most groups include
the use of structural elements within the sample itself to
align a sequence of images of the tomographic series or the
use of high-density markers, such as colloidal gold particles
with a diameter of 10
20 nm, as fiducial markers. In the first
case, the advantage is that there is no need for an additional
step in sample preparation. On the other hand, thicker
samples do not always have sufficient contrast (especially
when low doses are used) to perform a manual alignment. It
is also more efficient to rely on the use of fiducial markers
when local distortions on the sample (e.g., anisotropic
shrinking) occur.
A tilt series is aligned (usually by cross correlation) and
processed by specific tools in the software until the
reconstruction step, which generates the digital volume.
Different algorithms are available to perform this task,
including the ‘‘weighted back-projection‘‘ (WBP) (Rader-
macher et al., 1992), simultaneous iterative reconstruc-
tion technique (SIRT) (Sorzano et al., 2001), and
algebraic reconstruction techniques (ART) (Marabini
et al., 1997); SIRT and ART usually produce tomograms
with a smoother appearance when compared with WBP,
although with higher computational costs. The digital
volume can then be oriented to any direction, rotated
along any axis (x, y, z), and digitally sectioned at any
orientation. These sections in the tomogram are then
used to generate the digital 3D model in the same way
3D models are obtained in reconstruction from serial
sections. Electron tomography yields sections of higher
resolution when compared to reconstruction from serial
sections, which is generally reflected in a higher-quality
reconstruction (Fig. 5).
In addition to the correct application of acquisition and
alignment tools, the quality of tomograms is intrinsically
related to the preservation of the sample structure, which
may be significantly altered after chemical fixation and
subsequent sample preparation steps or result from
beam damage during acquisition. The use of quick-frozen
and freeze-substituted samples (Van Harreveld et al.,
1965; Steinbrecht, 1980; Steinbrecht and Müller, 1987;
Murk et al., 2003a; Murk et al., 2003b; McDonald and
Auer, 2006; Girard-Dias et al., 2012) or the application of
more elaborate techniques

such as quick-freezing fol-
lowed by analysis of frozen-hydrated specimens with the
use of special cryoholders and low-dose techniques (e.g.,
cryoeletrontomography) (Nicastro et al., 2000)

have al-
lowed for the characterization of 3D structures in their
close-to-native state, minimizing the interference and
loss of resolution resulting from conventional sample prep-
aration and imaging conditions (McEwen et al., 1995; Leis
et al., 2009).
Mol. Reprod. Dev. 82:530–547 (2015)
 THREE DIMENSIONAL RECONSTRUCTION BY ELECTRON MICROSCOPY

Figure 5. Electron tomography of organelles after quick-freezing and freeze substitution. A: Image showing the Golgi (G) cisternae (arrows). B and
C: 3D model of the Golgi. D: Image showing a multivesicular body (MB), with arrows indicating internal vesicles. E and F: 3D models of different
multivesicular bodies. Scale bars, 200 nm (A) and 100 nm (D). Adapted with permission from Girard-Dias et al., 2012.

Despite the use of relatively thick sections in electron
tomography, where all cell structures contained in the
volume may be imaged, the maximum thickness used is
frequently not sufficient to comprise the whole volume of
the structure or organelle under study. In this regard, an
alternative method to increase the total volume of the
sample analyzed is the combination of serial sectioning
with electron tomography, or ‘‘serial-electron tomogra-
phy’’ (Soto et al., 1994; Noske et al., 2008; Girard-Dias
et al., 2012; Toyooka and Kang, 2014). In this approach,
the thickness of the serial sections will be the same
typically used for tomography (0.2
0.5 mm). As in 3D
reconstruction of serial sections, it is also necessary to
identify different profiles of the same cell in sequential
sections. The fundamental difference is that instead of
acquiring single images of these profiles, tomographic
series (tilt series) are acquired for each section. The
steps of alignment and 3D reconstruction of a tomo-
graphic series are carried out for each acquisition and
are applied to each tomogram separately. The resulting
tomograms are then grouped according to their se-
quence, thus forming a single volume made up of several
sections. The volume of the structures to be studied is
thereby increased, and is now limited to the sum of the
thicknesses and number of the serial sections. The
volume of data (memory) generated in electron tomog-
raphy is proportional to the product of serial data volume
of a single section times the number of serial sections
used and the resolution of the acquired images. Thus,
the tomogram generated after the end of every joint
tomogram is usually a file with large data volume, and
therefore requires the use of computers with higher
processing capacity, especially for the segmentation
and rendering steps.
SCANNING ELECTRON MICROSCOPY-BASED
APPROACHES
In the recent years, 3D reconstruction techniques using a
scanning electron microscope (SEM) experienced a tremen-
dous advance. SEMs have long been used as classical
surface-characterization instruments. The interaction of
the electron beam with a sample surface generates a num-
ber of signals that can be used to form an image. Within a
SEM, an electron beam is scanned across a specific area on
the surface to be analyzed, and the signal (secondary
electrons, backscattered electrons, X-rays, Auger, and et-
cetera) collected from each scanned point by a specific
detector is represented as a gray value in an image pixel.
The intensity of the signal in the final image is dependent on
the surface topography. As the signals collected arise from
the surface of the sample (different depths depending on the
signal and the microscope setup), bulk samples can be
analyzed and details of the surface can be imaged (reviewed
by Bozzolla and Russel, 1992). To improve signal detection
and avoid charging effects, biological samples prepared for
conventional SEM analysis are usually coated with metals
such as gold, platinum, tungsten, or iridium.
In order to apply SEM-based methods to 3D reconstruc-
tion, data acquisition has to be performed in such a way that
sequential images of a flat surface are obtained. As the

Mol. Reprod. Dev. 82:530–547 (2015) 537

Molecular Reproduction & Development
detection of secondary and backscattered electron signals
from flat, embedded, and stained biological samples can
generate images that are very similar to those obtained with
a TEM, the sequential acquisition of images could provide
information that is very similar to serial sectioning with a
TEM. This is because stained regions (e.g., membranes,
nuclei) generate more signal (both secondary and back-
scattered electrons), resulting in an image that looks like a
TEM image with inverted contrast (reviewed in Kizilyaprak
et al., 2014a; Kizilyaprak et al., 2014b).
Improvements in signal detection methods, especially
using new detectors that have better sensitivity for low
energy electrons, have led to tremendous improvements
in image quality from SEMs. As a consequence, novel
strategies for the acquisition of serial images were devel-
oped, resulting in a number of SEM-based techniques.
These methods differ from TEM-based methods (3D re-
construction from actual serial sections) mainly due to
different approaches to sectioning and image acquisition
strategies; the subsequent image processing, segmenta-
tion, and reconstruction steps are very similar for the
different 3D reconstruction approaches that make use of
serial sections. The term ‘‘3D reconstruction from serial
sections’’ will thus be generally applied to the 3D recon-
struction techniques currently available, as discussed
below.
SEM images are formed by secondary and backscat-
tered electrons that emerge from the surface of bulk sam-
ples as they are scanned by an electron beam, therefore the
interaction volume between the incident electrons and the
sample becomes an issue. The higher the voltage, the
larger the interaction volume; the larger the interaction
volume, the lower the resolution. The lateral resolution is
therefore inversely proportional to the microscope voltage.
In addition, as secondary- and backscattered-electron sig-
nals come from different sample depths (secondary elec-
trons usually come from the regions closer to the surface
than backscattered electrons), the different signals may
provide both different contrast and surface detail (second-
ary-electron images tend to reveal more surface details,
such as ‘‘curtaining’’ effects). Images obtained at low vol-
tages generally provide better lateral resolution (due to the
smaller interaction volume), but at the cost of some signal.
In this case, the use of microscopes equipped with a field
emission gun

whose the beam has a smaller diameter
with more current density (brightness) then conventional
filaments

allows for the use of lower voltages (between 1
and 5 kV), which significantly improves the signal-to-noise
ratio while maintaining lateral resolution (Bozzolla and
Russel, 1992).
Recent Developments in Serial Sectioning: The
Use of SEM-Backscattered Imaging to
Reconstruct Larger Volumes
Obtaining 3D data from large sample volumes has been
one of the major obstacles in structural biology. During the
last decades, several approaches were developed to over-
come this limitation, with most of them seeking to access
538
MIRANDA ET AL.
larger volumes at high resolution. As discussed above,
classical 3D reconstruction by serial sectioning using a
TEM has the disadvantage of being a labor-intensive meth-
od that achieves small volumes due to the limited number of
sections that can be collected on an electron-microscopy
grid, unless special methods are applied (White et al., 1986;
Bumbarger et al., 2013). More sophisticated methods such
as electron tomography can achieve higher resolutions, but
usually require the use of intermediate- or high-voltage
microscopes for imaging larger volumes (thicker samples).
Alternative methods, such as serial block-face (SBF) and
focused ion beam (FIB) milling scanning electron micros-
copy (discussed below), are promising approaches since
they allow for faster reconstruction of large volumes with
resolution close to that obtained with a TEM. As occurs with
electron tomography, however, these approaches require
expensive and sophisticated equipment that are not yet
available in most laboratories.
A new approach, initially named ‘‘array tomography’’
(Micheva and Smith, 2007) and later ‘‘serial-section scan-
ning electron microscopy’’ (Horstmann et al., 2012), col-
lects the ribbons of ultra-thin sections, routinely prepared
by floating in the water at the diamond knife boat, onto a
hydrophylized silicon wafer that is mounted via forceps onto
a custom-built manual micromanipulator device attached to
the ultramicrotome. This procedure dramatically reduces
the handling of the sections, consequently avoiding errors
during data collection. Once on the silicon wafer, the
ribbons of ultra-thin sections can be dried and stained
with uranyl acetate and lead citrate. The silicon wafer is
then transferred to a SEM equipped with a field emission
gun, where backscattered electron signals are usually used
to form an image (Leighton, 1981; Denk and Horstmann,
2004) (Fig. 1C). Secondary electron signals can alterna-
tively be acquired, depending on the microscope configu-
ration and detectors available. To obtain a good signal-to-
noise ratio, the samples must be impregnated with heavy
metals (e.g., osmium tetroxide) since backscattered elec-
trons are produced by the elastic interaction of the primary
electrons from the beam with the nuclei of atoms in the
specimen. The signal-to-noise ratio is optimized because
the interaction of the electron beam with the exposed
surface generates more backscattered electrons from
the regions impregnated by the heavy metal (e.g., osmium
tetroxide-fixed membranes). The images generated are
analogous to TEM images of ultra-thin sections, but present
an inverted contrast (dense regions seen in a TEM image
due to elastic scattering are seen as bright areas) (Fig. 6A).
By digitally inverting the contrast of these images, one can
obtain images with similar features to those obtained by a
TEM (Fig. 6B), which, once aligned, can be used for 3D
reconstruction (Fig. 6C
F).
The major advantages of this serial-section SEM meth-
od are related to the sample preparation and the quality of
the images. As the sections are collected on the hydro-
phylized silicon wafers, they are firmly attached, preventing
the sections from folding upon themselves. In addition, as
the wafers are much larger than an electron microscope
grid, it is possible to collect a larger number of sections per
Mol. Reprod. Dev. 82:530–547 (2015)
 THREE DIMENSIONAL RECONSTRUCTION BY ELECTRON MICROSCOPY
Figure 6. 3D reconstruction using a microtome-based serial section within an SEM chamber. A: Backscattered electron image of a sea urchin egg.
B: inverted image of panel A. C
F: Automatic segmentation (isosurface) of internal structures (yolk granules) from the selected area in panel B.
Scale bars, 50 mm (A and B), 10 mm (C
E), and 5 mm (F).
ribbon. An alternative, improved method for automated
serial section collection directly on a tape, using a device
attached to the ultramicrotome, further reduces disruption
of the ribbon, thereby increasing the number of sections per
sample (Schalek et al., 2011). This process was named
automated tape-collection ultramicrotomy (ATUM). In both
cases, high-resolution images can be acquired with a
lateral pixel resolution of 3.7 nm, providing TEM-like im-
ages (not TEM-like resolution) of cellular structures for
subsequent segmentation and 3D reconstruction (Micheva
and Smith, 2007; Horstmann et al., 2012). Such ribbon-
collection approaches enables the acquisition of large-
volume data for tissues and cells, providing high-quality
images with the use of conventional sectioning equipment
and a field-emission SEM equipped with a backscattered-
electron detector. An additional advantage is that the whole
surface of the section can be scanned without the interfer-
ence of grid bars. But this imaging approach in general
achieves a lateral resolution slightly inferior to that obtained
by TEM tomography.
Serial Sectioning Within the Microscope
Chamber Using a SBF-SEM
A recent alternative tool for acquiring 3D information
over large areas and extensive volumes is the SBF-SEM
Mol. Reprod. Dev. 82:530–547 (2015)
(Fig. 1D), which sections the specimen via an ultramicro-
tome built inside the SEM chamber. Each newly exposed
face is imaged in sequential order, followed by making a
new section in an automated fashion (Leighton, 1981; Denk
and Horstmann, 2004). In an SBF-SEM system, samples
embedded in plastic resin (as is regularly prepared for a
TEM) are placed in the chamber of an adapted SEM. The
sample block face is then repeatedly sectioned with the use
of a diamond knife, and sequentially imaged. The proce-
dure for obtaining the serial images is fairly simple: the
ultramicrotome is attached to the door of the SEM, and can
be operated (for fine adjustments) from outside the cham-
ber. The sample is then adjusted with respect to the dia-
mond knife, the door is closed, and the chamber is
evacuated. The face of the block is positioned just below
the pole piece of the SEM. The diamond knife removes a
section from the surface, and then moves away so that the
freshly cut block face can be imaged for backscattered and/
or secondary electron signals. Settings are optimized for
the magnification, scanning speed, and resolution required,
after which the serial acquisition can take place automati-
cally. There is basically no limit to the volume that can be
sampled in the block

up to order of millimeters in some
cases (Wilke et al., 2013). Zeta resolution is limited by the
section thickness (down to 15
20 nm in a routine basis)
and the voltage used (Hughes et al., 2014), but it is usually
539
Molecular Reproduction & Development
lower than in FIB-SEM approaches (see below). Lateral
resolution is usually dependent on the imaging settings
(detectors, scanning speeds, and etcetera), and is also
influenced by the voltage used

which is similar to serial-
section SEM, ATUM, and FIB-SEM. The SBF-SEM system
can typically work overnight, and the raw images are
directly exported to software for obtaining stacks for sub-
sequent reconstruction, alignment, segmentation and
quantification, as mentioned above. The cut area can be
as large as that covered by the diamond knife; this feature
alone makes a SBF-SEM more attractive to the study of
large samples compared to the FIB milling approach,
whose imaging area is usually limited to the field of view
of the FIB column (see below) and is usually smaller than
diamond-knife-cutting surfaces.
The sample preparation procedure for a SBF-SEM is
very similar to those used for a regular TEM. Higher
image contrast is usually achieved when en block staining
with osmium tetroxide and uranyl acetate is performed
(Hughes et al., 2014). As mentioned above, freeze-sub-
stitution methods are also a good choice in this case
because of the general improvement in the preservation
of cellular architecture when samples are fixed by quick-
freezing methods. Resins like Durcupan (from Electron
Microscopy Sciences) have been successfully used to
generate satisfactory results from up to 3,000 serial
images and roughly 300 mm in the z-axis (unpublished
results).
3D Reconstruction With FIB-SEM
The dual-beam (or cross-beam) SEM is a combination of
an SEM, usually equipped with a field-emission source,
with an ion column (usually gallium) using a focused ion
beam (FIB) that coincides with an electron beam at the focal
point on a sample (Heymann et al., 2006). The interaction of
the electron as well as the ion beam with the sample
generates signals that are detected by backscattered or
secondary electron detectors, from which images are gen-
erated. The FIB is mounted at an angle of about 508 (usually
528
548, depending on the microscope manufacturer)
relative to the electron column (Fig. 1). In its original
conception, the interaction of the FIB with the sample
can be used to perform controlled micro-abrasions and
nano-prototyping modifications in specific areas, as depos-
its of metallic layers or hydrocarbons in specific regions of
the sample. Indeed, the use of FIB-SEM in the life sciences
started with its application on hard materials (teeth, bones,
insect exoskeletons) in order to control the exposure of
cavities (Langford, 2006). More recently, this methodology
has been applied as an alternative method for the 3D
reconstruction of large volumes with electron-microscopy
resolution.
The combination of imaging with an electron beam and
abrasion with the FIB in a dual-beam electron microscope
opened new possibilities for serial imaging and 3D recon-
struction (Merchan-Perez et al., 2009; Kizilyaprak et al.,
2014a; Kizilyaprak et al., 2014b). In a FIB-SEM, section-
540
MIRANDA ET AL.
ing occurs inside the microscope chamber (as in a SBF-
SEM) via the FIB, and signals from the newly exposed
surfaces are collected to form images. Serial sections
from resin-embedded samples submitted to osmium-fix-
ation and/or en block staining originally prepared for a
TEM are milled, while images from the electron beam
interaction are acquired afterwards in an automated fash-
ion. The sequence of cycles, named ‘‘slice and view’’
(‘‘slice’’, sectioning by the FIB; ‘‘view’’, imaging of back-
scattered electrons), results in a series of images for
subsequent 3D reconstruction, as in the other serial
section techniques (Bushby et al., 2011). Over the course
of the process, the sample (usually embedded in epoxide
resin) is initially protected with a layer of platinum (to avoid
excessive abrasion by the ion beam), and ion milled to
attain the necessary geometry to expose the surface from
which the images will be obtained. This surface is then
polished with the ion beam in order to produce a flat
surface to begin the ‘‘slice and view’’ cycle.
As the volume that can be sampled (or reconstructed)
is basically limited by the sample geometry in the resin, it
is possible to produce high-resolution models of large
volumes (tens of micrometers) with a FIB-SEM (Bushby
et al., 2011; Schneider et al., 2011; Villinger et al., 2012).
Furthermore, since the imaging and milling steps are
controlled by an automated system, the acquisition of full
volumes from several cells and organelles is much less
laborious, providing an easier way to perform morpho-
metric analysis with a number of cells

enough to apply
statistical tests to the 3D measurements (Medeiros et al.,
2012) (Fig. 7).
Specific sites of interest can also be pre-selected since
flat block surfaces previously smoothed by ultramicroto-
my may provide good backscattered (sometimes second-
ary) signals and allow the acquisition of low magnification
images (Hekking et al., 2009). With this method, sites of
interest can then be pre-selected and subsequently
zoomed in so that specific regions are imaged at high
resolution (Hekking et al., 2009). Aclar-embedded, fluo-
rescence-labeled cells may also be used for fluorescence
microscopy-3D FIB-SEM correlative studies (Jimenez
et al., 2010). In this case, Aclar pieces can be marked
either by needle engraving, grid printing using a carbon
coater (an electron-microscopy grid is placed over an
Aclar piece before coating, leaving a grid pattern after
removing the grid), or even FIB engraving before placing/
growing the cells. As Aclar is transparent, cells can be
imaged in the light microscope (using phase contrast,
differential-interference contrast, or fluorescence ap-
proaches) to annotate their positions. After aldehyde-
osmium fixation, the samples can be embedded in epox-
ide resin and left to polymerize at the appropriate temper-
ature. Aclar can then be lifted and peeled off the block,
leaving a surface that is completely smooth and contains
both the cells and engraving marks so that cells can be
milled (Jimenez et al., 2010).
While the use of a FIB-SEM

and the other SEM-
based techniques mentioned here

can generate infor-
mation for a large volume of one sample (many cells and/
Mol. Reprod. Dev. 82:530–547 (2015)
 THREE DIMENSIONAL RECONSTRUCTION BY ELECTRON MICROSCOPY

Figure 7. FIB-SEM tomography of mouse red blood cells infected with malaria parasites. Cells were chemically fixed and embedded in epoxide
resin and submitted to alternating cycles of FIB milling and imaging using backscattered electron signals. The section thickness was 100 nm, and
achieved using a 1 nA FIB current. Imaging was done using 2 kV and a backscatter detector for low-energy electrons. A and B display a virtual
section and 3D model of the same cell at different angles. Scale bar, 3 mm.

or larger areas), segmentation of the structures contained
in the larger volume as well as the rendering steps
required to obtain 3D models of them can be very labori-
ous and time-consuming. Furthermore, the milling area is
limited to the FIB field of view, which is usually smaller
than the maximum area sectioned by a diamond knife in
the SBF method.
Although possessing a better zeta resolution (5
7 nm,
depending on the section thickness and strategy used),
the lateral resolution of FIB-SEM imaging is similar to that
obtained with other SEM-based methods; the interaction
volume (mainly due to the voltages used) and the depth of
the detectable backscattered electron signals can further
limited resolution. Given these limitations, SEM-based
methods are often classified as tools for intermediate
resolution (Fig. 1), between that of TEM-based serial
sectioning and electron tomography. Nevertheless, lip-
id-bilayer resolution in a SEM has been achieved using a
combination of FIB milling and secondary electron imag-
ing, which usually derive from shallower regions than
backscattered electrons, using quick-frozen and freeze-
substituted samples (Villinger et al., 2012). Further, the
use of FIBs assembled into cryo-SEMs (where adapted
cryostages allow for the observation of samples at low
temperature) has tremendously advanced the scope of
sample preparation for cryoelectron tomography (Marko
et al., 2006; Edwards et al., 2009; Hayles et al., 2010;
Rigort et al., 2010). Recent work has shown that FIB
milling can be performed under cryoconditions to micro-
machine a sample and produce thin layers of eukaryotic
cells in a frozen-hydrated state (Rigort et al., 2012).
These layers were compatible with cryosection thick-
nesses obtained by cryoultramicrotomy, and presented
some advantages, such as the lack of micro-fractures and
compression effects usually seen in sections obtained by
cryoultramicrotomy (Al-Amoudi et al., 2005; Pierson et al.,
2011).
SOFTWARE FOR THE ALIGNMENT AND
GENERATION OF A 3D MODEL
Advances in the computational field have brought sev-
eral improvements that benefit the 3D reconstruction pro-
cess. Most significantly, the development of machines with
a large capacity for data processing and the availability of
software for 3D imaging have increasingly expanded, pro-
viding sophisticated functionality while reducing required
manipulation of the data by the user, and generating more
accurate models and measurements. Several research
groups and companies are dedicated to the development
of 3D-imaging software, which makes variety of tools avail-
able to a user. Here, we briefly list some of the software
currently used by groups interested in 3D image
processing:
 IMOD: Developed at the Boulder Laboratory for 3-D Recon-
struction at the University of Colorado (http://bio3d.colo-
rado.edu/imod/), IMOD is a free package that contains
tools for image processing, modeling, and visualization
(Kremer et al., 1996). This is a powerful tool for the alignment
and reconstruction of tomograms

probably the most-used
software by many groups. IMOD has a user-friendly inter-
face and manuals that are very useful for beginners.
 Cytoseg: Developed in the National Center for Microscopy
and Image Research at the University of California, San
Diego (http://ncmir.ucsd.edu/), Cytoseg is free software for
automatic segmentation and visualization of 3D biological
datasets (Giuly et al., 2012). Cytoseg uses three steps of
automatic processing based on different parameters, mak-
ing it effective for automatic segmentation

which is more
accurate when used to reconstruct globular structures.
Cytoseg is a command-line-based tool.
 Tomosegmem: Developed by a group assembled from
different institutions in Spain (http://www.cnb.csic.es/

Mol. Reprod. Dev. 82:530–547 (2015) 541

Molecular Reproduction & Development
jjfernandez/tomosegmem), Tomosegmem is a free pack-
age that assists users in segmenting membranes present in
tomograms (Martinez-Sanchez et al., 2011). This software
was developed especially to segment membranes based on
the Gaussian-like membrane model. Tomosegmem is a
command-line-based tool, and visualization of the results
needs to be done using other software.
 Synapse Web: Developed in the Center for Learning and
Memory at the University of Texas at Austin (http://synap-
ses.clm.utexas.edu/tools/index.stm), Synapse Web hosts
different free tools that are able to do alignment, segmenta-
tion, and rendering (Fiala, 2005). Among the different soft-
ware packages available, ‘‘Reconstruct’’ is the most
complete because it incorporates tools for alignment and
segmentation beyond other functions. This software is
based on the Windows platform.
 Amira and Avizo Fire: Developed by Visualization Scien-
ces Group and FEI Company (http://www.vsg3d.com/),
Amira and Avizo Fire are commercial packages with tools
for image processing, modeling, and visualization. This
software has a user-friendly interface and powerful tools
for automatic and manual segmentation. Like any com-
mercial software, however, it requires substantial invest-
ment, which is not always the first choice for beginners.
 TomoJ: Developed by Sergio Marco’s group at the Institute
Curie (Paris, France) (Messaoudil et al., 2007), TomoJ is a
plugin for the alignment and reconstruction of tomographic
series in the ImageJ platform (National Institutes of Health,
imagej.nih.gov/ij/). TomoJ can be freely downloaded (http://
rsb.info.nih.gov/ij/download.html) and connected to a running
ImageJ program. The software has a user-friendly interface,
and the reconstruction of tomograms can be done using
different algorithms, including SIRT, ART, and WBP.
3D ELECTRON MICROSCOPY FOR GENERAL
BIOLOGICAL QUESTIONS
There is no general rule defining which type of instru-
ment or technique should be used to answer a specific
question. More and more methods are becoming available,
each presenting advantages and limitations regarding the
resolution of the data set, sample area, and volume to be
analyzed

which are usually the main factors to be con-
sidered when a decision to use one or other method has to
be made (see Table 1).
In general, high resolution is achieved at the cost of
volume in tomography, whereas high volumes are
achieved with SEM-based methods at the cost of resolu-
tion. For instance, if one intends to study small structures
(e.g., viruses, proteins or aggregates, isolated organelles,
intracellular structures, membrane contents within or out-
side the cell, interaction between organelles, and docking of
cytoskeleton elements), imaging large sample areas and
volumes is ‘‘usually’’ not an issue, so electron tomography
is normally the method of choice. If the desired volume
542
MIRANDA ET AL.
slightly exceeds the limit established for tomography (some
of them may exceed the section thickness used for tomog-
raphy, which will also depend on the voltage of the micro-
scope), tomography would still be the method of choice


particularly if this technique is coupled to serial sectioning
(e.g., 5 mm volumes) because larger reconstructed vol-
umes with tomography resolution will be generated (Peddie
and Collinson, 2014). Conversely, for large samples (e.g.,
whole cells, tissues, and embryos), TEM tomography is
‘‘usually’’ not the easiest way to go since imaging such high
volumes in a TEM would tremendously compromise reso-
lution. On the other hand, manual serial-section TEM
approaches would be very time consuming and labor in-
tensive. Other approaches, such as the SEM-based meth-
ods (e.g., ATUM [Schalek et al., 2011]), are better suited for
large-sample and -volume acquisitions. FIB-SEM and
SBF-SEM, which use automation for sectioning and imag-
ing, would be less laborious, albeit more expensive, than
manual serial sectioning.
Although microscope resolution has had tremendous
improvement with the design of new columns and detec-
tors, high-resolution imaging of large areas remains a
problem since the information is usually stored in an image
with limited resolution (e.g., 1000  1000, 3000  3000
pixels). A 1000  1000-pixel image of an area of
100  100 mm would give a pixel size of 100 nm. To
cope with that scaling, alternative approaches that combine
high-resolution imaging and large areas are available, and
provide impressive results

although they also require an
impressive amount of time and processing power because
they involve automatically stitching individual high-resolu-
tion, high-magnification images together to form a large,
high-resolution image. A few commercially available soft-
ware packages include stitching options that integrate with
the microscope, which together can acquire images up to
105,000  102,000 pixels (10 gigapixels). When com-
bined in a 3D matrix (after FIB milling, SBF, and etcetera),
this post-acquisition processing provides high-resolution
information of large areas and large volumes. Although a
very attractive and informative option, 3D information of
such large volumes at a high resolution encounters the
obstacle of dealing with such a tremendous amount of data.
Regardless of their specificities, all the methods have
been shown to give a particular contribution to the field.
Although guidelines such as those given above are avail-
able, the method chosen should ultimately be selected with
the assistance of a specialist in the field.
CONCLUDING REMARKS
The application of 3D reconstruction techniques in
electron microscopy has expanded tremendously over
the recent years. A number of techniques are available
to generate volumetric ultrastructural models, and com-
bination of a variety of strategies is now possible for
tailoring to specific biological questions and applications.
These options provide versatility to what was once a very
limited technique that was restricted to a few electron-
Mol. Reprod. Dev. 82:530–547 (2015)
 THREE DIMENSIONAL RECONSTRUCTION BY ELECTRON MICROSCOPY

TABLE 1. Key Features of Different Electron Microscopy Methods for 3D Studies

Time
Method Resolution Volume (for 1-mm thick sample) Equipment

Serial-sectioning Low resolution in x-, y-, Usually small volumes, A few hours. Time Ultramicrotome and a
TEM and z-axes (due to although intermediate -consuming and conventional TEM
the section thickness) volumes can be laborious. A number
obtained when serial of manual
sections are collected adjustments needed.
in different grids Profiles of the
structure of interest in
each section have to
be selected by the
user and
subsequently imaged
during the whole
process
Electron High resolution in x-, y-, Small volumes A few hours. (S)TEM with automated
tomography and z-axes (sample thickness Tomograms of serial tomography
(considering tilt- limited to 200
400 nm, sections can be capability
series and z- for a 200 kV TEM, grouped in a single
projection for example) tomography run
approaches, the (batch tomography).
resolution is lower Automated method
than obtained with
single-particle
approaches)
Serial-sectioning Intermediate resolution Large areas and A few hours. Time- SEM equipped with a
SEM or ATUM in x-, y-, and volumes can be consuming and l field emission gun
z-axes. Lipid bilayers can obtained (a large aborious. Profiles of and a backscattered
theoretically be seen number of sections the structure of electron (preferred)
in special cases can be placed in the interest in each or secondary electron
same silicon wafer). section have to be detector
The area is usually selected by the user
larger then in FIB- and subsequently
SEM imaged during the
whole process
SBF-SEM Intermediate resolution Large areas (usually Several hours. No need SEM equipped with a
in x-, y-, and z-axes. larger than in the FIB to select individual field emission gun
Lipid bilayers can milling methods) and profiles of the same and a backscattered
theoretically be seen volumes can cell; the cell is electron (preferred)
in special cases be obtained sequentially sectioned and or secondary
imaged in an electron detector and
automated fashion an ultramicrotome
into the chamber
FIB-SEM Intermediate resolution Large areas (usually Several hours. No need Dual-beam or Cross-
in x-, y-, and z-axes smaller than in SBF to select individual beam SEMs
(the z resolution can methods) volumes profiles of the same (FIB þ SEM
be slightly better than can be obtained cell; the cell is columns)
the other SEM-based sequentially
methods).Lipid- sectioned and
bilayer resolution has imaged in an
been demonstrated automated fashion

microscopy labs. Yet, novel approaches are still being
developed, and some of them may be of special interest to

such as obtaining correlative information
cell biologists

between light (fluorescence) and 3D electron microscopy.
This dual-purpose method can be applied to both TEM-
and SEM-based reconstruction methods, and can be
done in a number of different ways to ensure that correct
coordinates are assessed by both microscopy methods.
For example, beacons can be created by using known
marks on the sample substrate/resin block that are rec-
ognized under the light and electron microscopes; by
saving specific sample coordinates that are shared by
both a light microscope and a SEM; by using autocorre-
lation algorithms that find specific frequencies in the
Fourier space; and/or by observing a fluorescent sample
via a fluorescence microscope attached to a TEM column.
These correlation methods could have a tremendous
impact on cell biology

especially now with the

Mol. Reprod. Dev. 82:530–547 (2015) 543

Molecular Reproduction & Development
development of super-resolution light microscopy sys-
tems. When combined with new strategies for the visuali-
zation of large areas at high resolution, such multi-
dimensional approaches have the potential to strongly
impact our understanding of the 3D ultrastructural orga-
nization of biological systems at a high resolution.
ACKNOWLEDGMENTS
This work was supported by grants from the following
Brazilian agencies: Conselho Nacional de Desenvolvi-
mento Cient
ıfico e Tecnolo
gico (CNPq - Universal
480184/2012-7, 449256/2014-6, INCT em Biologia Estru-
tural e Bioimagem); Fundaa~o Carlos Chagas Filho de
Amparo Pesquisa de Estado do Rio de Janeiro (FAPERJ
-Programa Nœcleos Emergentes # E-26/111.185/2011);
Coordenaa ~o de Aperfeioamento do Pessoal de N
ıvel
Superior (CAPES); Financiadora de Estudos e Projetos
(FINEP).
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