Micron 42 (2011) 175–185

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Micron
journal homepage: www.elsevier.com/locate/micron

Review

Low voltage high-resolution SEM (LVHRSEM) for biological structural and

molecular analysis
Heide Schatten ∗
Department of Veterinary Pathobiology, University of Missouri, 1600 E Rollins Street, Columbia, MO 65211, USA

a r t i c l e i n f o a b s t r a c t

Article history: High-resolution scanning electron microscopy (HRSEM) is being used increasingly to gain new insights
Received 1 April 2010 into three-dimensional organization of biological structure, macromolecular complexes and interactions
Received in revised form 22 August 2010 of cellular components as well as isolated cell organelles. Modern scanning electron microscopes (SEMs)
Accepted 23 August 2010
combined with adequate sample preparation can now provide resolution comparable with that achieved
using transmission electron microscopes (TEMs) down to 2–5 nm for biological material. The versatility
Keywords:
of the instrument and new sample preparation techniques have allowed detailed analysis of chromo-
High-resolution scanning electron
somes, cytoskeletal components, virus and other biological material that has not been possible with TEM.
microscopy (HRSEM)
Resolution
The present review addresses resolution and specific specimen preparations for HRSEM, and highlights
Specimen preparation the importance of specimen preparation and choice of methods to achieve optimal results for proteins,
Cytoskeleton macromolecular complexes and subcellular structures using low voltage HRSEM (LVHRSEM).
Cell structure © 2010 Published by Elsevier Ltd.
Virus

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
2. Resolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
3. General sample preparation for SEM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
3.1. Conventional sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
3.2. Freezing methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178
4. Specific sample preparations for specific structural and macromolecular analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
4.1. Chromosome structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
4.2. Isolated mitotic spindles, centrosomes, and nuclear envelopes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
4.3. De-embedding of Epon-embedded thick sections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
5. Future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184

1. Introduction

The appreciation for scanning electron microscopy (SEM) has Maconnachie, 1979) caused by ethanol dehydration and inade-
increased significantly in recent years with the recognition that quate critical point drying have been overcome by several refined
modern SEMs combined with adequate sample preparation can preparation methods, as well as cryofixation to remove unbound
provide resolution comparable with that achieved using trans- or extracellular water from the specimen while preserving bound
mission electron microscopes (TEMs). Furthermore, the versatility water to retain structural and molecular integrity (reviewed in
of the instrument and sample preparation resulted in multiple Erlandsen, 2008).
new approaches to utilize SEM for molecular and structural imag- The present review addresses resolution and specific speci-
ing. Early preparation techniques associated with shrinkage in men preparations for LVHRFESEM. It highlights the importance
cell dimensions and collapse of molecular structure (Boyde and of adequate specimen preparation and the choice of meth-
ods required to achieve optimal results for specific analyses of
proteins, macromolecular complexes and subcellular structures
∗ Corresponding author. Tel.: +1 573 882 2396; fax: +1 573 884 5414. using low voltage high-resolution field-emission scanning electron
E-mail address: SchattenH@missouri.edu. microscopy (LVHRFESEM).

0968-4328/$ – see front matter © 2010 Published by Elsevier Ltd.

doi:10.1016/j.micron.2010.08.008
176 H. Schatten / Micron 42 (2011) 175–185
2. Resolution
The limitations of spatial resolution for biological samples in the
SEM has been defined by Joy and Pawley (1992) as follows: “The
spatial resolution of the scanning electron microscope is limited by
at least three factors: the diameter of the electron probe, the size
and shape of the beam/specimen interaction volume with the solid
for the mode of imaging employed, and the Poisson statistics of the
detected signal.” Enormous progress in instrumentation and speci-
men preparation development has moved SEM in close competition
to TEM, achieving resolution of biological material down to the
2–5 nm range and in some instances down to 1 nm. Improved reso-
lution has allowed gaining new knowledge on biological structure,
macromolecular complexes, and cellular interactions of intracel-
lular components, cell–matrix interactions, and isolated biological
material. However, there is no universal answer to the question on
resolution as it depends on a number of different factors and there
is no standard method of measuring SEM resolution despite the
search for an ideal bulk SEM test specimen (reviewed in Pawley,
2008; McMullan, 2008).
The past two decades have seen significant improvements in
resolving power of commercially available SEMs as a result of
several developments including the use of field-emission cath-
odes as electron sources (Crewe et al., 1968) and improved lens
designs (Pawley, 1992). The primary beam diameter of modern
high-resolution SEMs is less than 1 nm (Nagatani et al., 1987). Spec-
imen preparation is still one of the challenging components in
achieving optimal resolution as will be discussed in Section 3. As
has been discussed by Walther (2008), field-emission SEMs of the
in-lens-type offer the smallest available probe size of less than 1 nm
at V0 = 30 kV and about 1 nm at V0 = 10 kV (Nagatani et al., 1987). At
1 kV, however, the diameter increases and reaches about 3 nm at
1 kV (Nagatani et al., 1987).
Several excellent articles have addressed the various fac-
tors affecting resolution in LVFESEM (reviewed in Pawley, 2008;
Erlandsen, 2008; Walther, 2008, and others). The history of SEM
development and improvement in resolution has been excellently
reviewed by McMullan (2008) and progress has been remarkable
since 1960 when a resolution of 200 nm at 1 keV was considered
excellent. The development of ultra-high vacuum technology as
well as corrections for aberrations played a critical role in allowing
SEMs to be marketed (reviewed in Joy, 2008a,b; McMullan, 2008;
Newbury, 2008) and overcome setbacks that had been experienced
up to the middle 1960s. As mentioned above, by now the practi-
cal resolution of LVSEM rivals that obtained with thin-section TEM
which has yielded three-dimensional new information in stud-
ies of cell structure; an additional advantage of using LVFESEM
includes detailed structural information with high spatial resolu-
tion.
The early SEM instruments developed by the Oatley group
around 1950 (Oatley, 1982; reviewed by Pawley, 2008) used
hot tungsten filaments and were operated at 10–30 kV, although
Thornley used low V0 to avoid charging when using uncoated,
frozen biological samples (Thornley, 1960; Thornley and Cartz,
1962). In the 1970s, LaB6 Schottky thermionic cathodes (Broers,
1974) and cold field-emission (FE) cathodes (Crewe et al., 1968;
Crewe, 1973; Hainfeld, 1977) were introduced for SEM use. Further
developments included using a TEM-type objective lens allow-
ing collection of secondary electrons (SEs) and backscattered
electrons (BSEs) from a specimen mounted inside the lens field
which reduced lens-aberration coefficients by a factor of up to
20 (reviewed in Pawley, 2008). Later instrument improvements
allowed FESEM performance below the lens which is now used
in most modern FESEMs. Improvement in SE and BSE detectors
followed in subsequent years and the design of an improved
YAG/BSE detector (Autrata, 1990) allowed signal collection from
gold particles (reviewed in detail by Pawley, 2008) that was one
of the keys to reliably detect immuno-labeled particles associated
with biological structure without steric interference that otherwise
oftentimes affects correct analysis of immuno-stained molecular
components (reviewed in Albrecht and Meyer, 2008). Analysis
of immuno-labeled structures can either be achieved by using
different voltages and overlay of separate images (for example,
small gold particles have been detected at 5 kV while detection
of biological structure utilized 20 kV (Pawley and Albrecht, 1988;
reviewed in Pawley, 2008)), or by employing a combination of
BSE and SE recording simultaneously (reviewed in Hermann et al.,
1996).
The high resolution available with LVFESEM has also allowed
more detailed immunolabeling of biological structure as detailed by
Albrecht and Meyer (2008). Small metal labels as small as 2–3 nm
can be imaged on very thinly coated or completely uncoated spec-
imen.
Taken together, the development of electron sources, lenses,
vacuum systems and magnetic shielding considerably contributed
to high-resolution LVSEM (reviewed by Pawley, 2008). Instrument
development in 1986 included an FE source, a short focal-length
lens, and a good vacuum system that resulted in achieving res-
olution down to 3 nm at 1.5 kV and provided the long-desired
opportunity to use LVSEM for high-resolution topographic imaging.
As mentioned above, it is now possible to use in-lens instruments
to achieve resolution of 1 nm at 1 kV and instruments in which the
specimen is mounted below the final lens to achieve resolution of
1.5 nm. The Hitachi S-900 at the Integrated Microscopy Resource
in Madison, WI, was the first instrument to offer such high reso-
lution afforded by then important instrument modifications and
thorough evaluation of sample preparations. This instrument in an
NIH-funded resource environment has served numerous investiga-
tors involved in cutting-edge research (reviewed in Pawley, 2008)
offering ca. 5× better resolution than had been possible previously
(reviewed in Pawley, 2008). Several images obtained on this instru-
ment are shown in the present review paper. All manufacturers
of SEM instruments now offer capabilities that allow resolutions
down to 3–5 nm or even 1.5 nm under favorable specimen prepa-
ration conditions using below the objective lens specimen imaging.
Modern high-resolution SEMs allow 3 nm resolution at any V0
above 1.5 kV. It has to be stressed that specimen preparation is
crucially important to obtain high-resolution image results; the
best instrumentation is only as good as the best specimen and
lack of expertise may affect the results significantly. Therefore, a
major part of the present review is dedicated to specimen prepa-
ration.
3. General sample preparation for SEM
The importance of adequate sample preparation cannot be
overemphasized, as the best instrumentation is only as good as the
sample and the best instrumentation cannot compensate for poor
specimen quality. Maintaining structural integrity is an absolute
requirement for specimen preparation and subsequent analysis
in the vacuum of the microscope. A variety of different sample
preparation techniques are available that have been elaborated by
various investigators. In this section, the most common specimen
preparation techniques are discussed. Specialized sample prepa-
ration techniques that had been worked out for specific research
applications are featured in Section 4.
3.1. Conventional sample preparation
A typical protocol includes fixing the biological sample with
appropriate aldehydes such as 2% paraformaldehyde/2% glu-
 H. Schatten / Micron 42 (2011) 175–185
Fig. 1. Actin filament displaying helical twist 5 nm subunits of in vitro preparations
after dilute glutaraldehyde and cryofixation. Bar = 60 nm.
Reprinted from Erlandsen (2008).
taraldehyde in 0.1 M phosphate buffer (PBS) followed by PBS rinses,
postfixation in 1% osmium tetroxide in 0.1% PBS, rinses in PBS,
dehydration in increasing series of ethanol or acetone, critical point
drying, mounting on aluminum stubs, and coating.
Chemical fixation is adequate for many samples but, depending
on the questions addressed, it is important to remember that chem-
ical fixation may destroy structural integrity of some structures of
interest. For example, glutaraldehyde cross-links the free amino
groups of polypeptides and amino acids and inactivates enzymes. It
is therefore inadequate for enzyme localization and related studies.
Dehydration is being used routinely to prepare samples for
viewing in a vacuum environment. However, dehydration may
be damaging to biological structures because the shape of a
macromolecule or membrane is produced and maintained by its
interactions with water which may be destroyed by removing
water during the dehydration process. As much as 30–60% shrink-
age has been reported after preparing soft tissue for SEM (Boyde and
Fig. 2. Spread human platelets after light fixation in glutaraldehyde followed by
immunolabeling for cell-adhesion molecules and cryofixation. (A) Shows Low mag-
nification of the platelet while (B) shows high-magnification of dimeric P-selectin.
(C) Displays gold-labeled GPIb in the GPI-IX complex; (D) reveals gold-labeling
of individual integrin molecules for GPIIbIIIa. Bars A = 1 ␮m; B = 130 nm; C and
D = 100 nm.
Reprinted from Erlandsen (2008); originally published in Erlandsen et al. (2001).
177
Fig. 3. Stereo-pair of 3T3 cell after fresh-freezing in propane, freeze-fracture and
thawing into fixative followed by critical point drying and coating with ion-
beam sputtered Pt. Nucleus (top), nuclear membrane and peri-nuclear space are
clearly discernable. Image provided by Haggis (Agriculture Canada, Ottawa). Field
width = 2.8 ␮m.
Reprinted from Pawley (2008).
Maconnachie, 1979, 1981). However, large macromolecules and
structural components associated with them or covalently attached
are preserved (Ris, 1985, 1988, 1990, 1991).
Critical point drying (CPD) is one of the preparation procedures
requiring several precautions to avoid artifacts (Ris, 1985). Traces
of water contaminating the intermediate liquid used for drying
(ethanol or acetone) or in the liquid CO2 transition fluid may distort
ultrastructure and induce artifacts which has clearly been shown
by Ris (1985) in a thorough series of elaborate control experiments.
In this case, studies clearly showed that the previously termed
microtrabecular structure was the result of residual water arti-
facts and not a newly discovered structure (Ris, 1985). This study
has been critically important to raise awareness of facts and arti-
facts and again, it needs to be stressed that the most impressive
instrumentation cannot compensate for inferior or improper sam-
ple preparation. Thorough CPD is one of the critical aspects for
producing artifact-free samples.
Coating is important for biological structure, as most biologi-
cal samples have insulating characteristics and need to be made
more conductive. Typically, a thin metal coating is applied to reduce
the effects of charging, e.g. glaring on the sample. The coating also
helps that secondary electrons from the very surface of the sample
Fig. 4. The importance of adequate specimen preparation is explained in this TEM
image of bacteriophage T4 polyheads. While fixation by conventional methods and
critical point drying (A) does not preserve substructure, cryo-immobilized and par-
tially freeze-dried samples show easily discernible subunits (B). Bar = 100 nm.
Reprinted from Erlandsen (2008); originally published in Hermann and Müller
(1991).
178 H. Schatten / Micron 42 (2011) 175–185

(from the coating layer) only are released, whereas on uncoated
biological samples secondary electrons can also escape from a cer-
tain depth, reducing surface resolution (discussed in Hermann and
Müller, 1991). Heavy metals or heavy metal compounds are used
for coating; however, it is oftentimes difficult to determine the cor-
rect amount of coating which eliminates charging, but also allows
reliable viewing of biological structures without obscuring the
areas of interest. Coating may affect biologically relevant resolution
(reviewed in Pawley, 2008). For LVFESEM, 1–2 nm gold–palladium
applied by sputter coating has been optimal for a large variety
of samples (reviewed in Schatten, 2008). Optimizing the coating
thickness may be required for delicate biological structure.
A variety of coating procedures have been described and dis-
cussed in the literature (Peters, 1980; Peters, 1982, 1985, 1986a,b,
1988; Peters and Fox, 1990) offering several excellent alternative
methods which include coating with a uniform film of Ta or Cr
deposited as a 1–2 nm layer by Penning sputtering before view-
ing with 30–40 kV. Fine details below 5 nm have been observed
with this method. Others have used Pt, W, and Ta by DC-ion
sputtering to view cells growing on EM grids (Bell et al., 1989;
Lindroth et al., 1988; Lindroth and Sundgren, 1989). For biological
samples with charging problems, Walther and Hentschel (1989)
developed a double-coating technique by which the sample is
first coated with a thin layer of heavy metal (platinum–carbon
or tungsten with an average thickness of 2–3 nm) followed by a
5–10 nm carbon layer to improve mechanical stability and enhance
electrical conductivity (Walther, 2008). These samples are best
imaged using the backscattered electron signal, since then con-
trast formation occurs at the thin heavy metal layer, which is in
close contact with the biological structure of interest, whereas
the secondary electron signal would image the overlaying carbon
layer.
Charging (e.g. glaring on the sample) may occur more often
on flat specimens, as more electrons accumulate at the sur-
face while fewer leave the surface which may especially occur
when viewing samples at 5–20 kV (reviewed by Pawley, 2008).
Although it is oftentimes advertised that no coating is neces-
sary under specific instrument conditions such as low voltage
(LV) or environmental (ESEM), charging oftentimes does occur
and a thin layer of coating may still be required to avoid charg-
ing.
Good sample preparation and importantly, the investigator’s
expertise are among the most important criteria for reliable results
using EM.
3.2. Freezing methods
Ultrarapid freezing is oftentimes used to provide superior
ultrastructure and to preserve molecules in a more native state
compared to chemical fixation. Ultrarapid freezing typically is
achieved at a rate of 104 –105 ◦ C/s to avoid formation of damaging
ice crystals in cells or tissue. At this temperature rate, cellular water

Fig. 5. SEM image using backscattered electrons (BSE) shows high-pressure frozen and cryofractured pancreas tissue displaying porous structures in the ER membranes
(arrows). (a) A perpendicular fracture to the ER membrane (center) and fracture parallel to the membranes (right). (b) and (c) Higher magnifications displaying ribosomes
and filamentous structures. The smooth extraplasmic fracture face (EF) faces the ER lumen; the rough protoplasmic fracture face (PF) faces the cytosol.
Reprinted from Walther (2008).
 H. Schatten / Micron 42 (2011) 175–185
Fig. 6. High-resolution cryo-SEM image using secondary electrons (SE) shows ade-
novirus at high magnification.
Reprinted from Walther (2008).
becomes vitrified rather than forming ice crystals. Commonly used
freezing methods include the following.
Plunge freezing requires plunging a specimen into a liquid cryo-
gen such as supercooled liquid nitrogen or supercooled ethane
or propane which allows an average depth of vitrification of ca.
1–2 ␮m with minimal ice crystal artifacts.
Slam freezing (cold metal block freezing) is achieved by slam-
ming a specimen onto a copper or silver block that has been chilled
to −196 to −269 ◦ C with liquid nitrogen or liquid helium, allowing
an average depth of vitrification of ca. 10–15 ␮m with minimal ice
crystal artifacts.
Propane jet freezing is achieved by sandwiching a 200–500 ␮m
thick specimen between two metal plates that are clamped into a
device that directs jets of liquid propane cooled with liquid nitrogen
against both sides of the specimen plates; this allows an average
depth of vitrification of ca. 40 ␮m with minimal ice crystal artifacts.
High-pressure freezing follows propane jet freezing or liquid
nitrogen and is achieved by pressurizing the specimen to 2100
atmospheres to suppress or reduce growth of ice crystals at the
moderate freezing rates that can be achieved in the depth of the
sample. High pressure lowers the freezing point of water as well
as the rate of ice crystal formation, allowing an average depth of
vitrification of ca. 500 ␮m with minimal ice crystal artifacts.
Several variations of these basic freezing methods have been
applied to various specimens, some of which will be described
below. Freezing followed by freeze-drying has been used suc-
cessfully (Pawley and Ris, 1987) and freezing followed by
freeze-substitution is one of the methods that has gained increasing
popularity (Erlandsen, 2008). Direct observation of frozen speci-
Fig. 7. High-resolution cryo-SEM image using secondary electrons (SE) shows freeze-dried and tungsten-coated actin filaments in which actin subunits are directly visible.
Reprinted from Walther (2008).
179
Fig. 8. Cryo-FESEM images of the outer nuclear membrane (o) of mock-infected (A)
and HSV-1-infected (B) HeLa cells after 10 h of incubation. (A) Nuclear pores appear
as round, small depressions with a distinct NPC structure, and a few appear as small
buttons (arrows). (B) Pores appear either as small but deep depressions without
obvious NPC structure or as buttons. Some of them have larger diameters and are
over top the nuclear membrane (arrows). In addition, there are holes with distinct
borders (arrowheads) and numerous budding capsids. The outer nuclear membrane
is locally broken away, giving view to the inner nuclear membrane (i). Bars = 200 nm.
Reprinted from Wild et al. (2009).
mens (Pawley et al., 1991) has provided somewhat better results
regarding resolution (better than 3 nm) compared to freezing fol-
lowed by freeze-drying. Freeze-fracture (Haggis and Pawley, 1988)
is an excellent method to view intracellular structures which has
been reviewed by Pawley (2008). Dry fracture of tissue culture cells
has been achieved by touching intact cells to the surface of adhesive
tape (Ris, 1988, 1989; Lim et al., 1987; Ris and Pawley, 1989) which
allows excellent insights into intracellular structure. This method
may also be useful to view incorporation of labels that decorate
internal cell structure. The cell surface can be viewed first before
removing it for viewing the internal cell structure.
Cryo-SEM of chemically fixed cells has been described by
Erlandsen (2008). As mentioned above, several cryo-preparation
methods are available to arrest cells in a “life-like” state (Mueller,
180 H. Schatten / Micron 42 (2011) 175–185

Fig. 9. Scanning electron micrograph of plant chromosomes in metaphase (top image; barley), early prometaphase (lower left image; barley) and prometaphase (lower right
image; rye) isolated with the drop/cryo technique. In metaphase the chromatids exhibit numerous chromomeres (top image; circle) and begin to separate. The centromere
(top image, arrow) is characterized by exposed parallel fibers. Parallel fibers (lower left and right image; arrows) and chromomeres of different sizes (lower left and right
image; circles) are seen in all stages of condensation.
Lower right image from Zoller et al. (2004); reproduced by permission of S. Karger AG, Basel. Entire figure reprinted from Wanner and Schroeder-Reiter (2008).

1990). Cryo-immobilization occurs in milliseconds compared to
chemical fixation which may take seconds or even minutes.
Freeze-substitution in acetone, methanol or other solvents can be
used for subsequent processing and permanent fixation. In some
cases, chemical fixation with low concentrations of glutaralde-
hyde (0.1–1.0%) is used for 10–15 min to stabilize macromolecules
prior to cryo-immobilization (Chen et al., 1995). Such prepara-
tion methods have been used for the analysis of macromolecular
complexes and resulted in preserving remarkable detail of actin
filaments (Erlandsen, 2008) after coating with chromium. The heli-
cal twists of two polypeptide chains in the filament and 5 nm
subunits are clearly seen in Fig. 1. Other studies of macromolecu-
lar complexes revealed biological resolution of 2–3 nm. Erlandsen
et al. (2001) used cryo-methods to study details of the glyco-

Fig. 10. Stereomicrographs of barley metaphase chromosomes at different stages of artificial decondensation (fixation with glutaraldehyde and treatment with proteinase
K). During stretching, chromomeres loosen and can be recognized as bundles of solenoid fibers. The architecture of the centromere also loosens, showing three-dimensional
arrangement of parallel matrix fibers (A). After ultimate stretching, the number of matrix fibers is decreased and the chromomeres are stretched and decondensed to the
level of the individual solenoid fibers (B, arrow).
Wanner and Formanek, 2000; reproduced by permission of Elsevier. Partial figure reprinted from Wanner and Schroeder-Reiter (2008).
 H. Schatten / Micron 42 (2011) 175–185 181

Fig. 11. Isolated mitotic apparatus of dividing sea urchin cells after critical point drying displaying microtubules and associated substructures observed at 1.5 kV without
charging artefacts after fracturing and light coating.
Image from collaborations (Thompson-Coffe et al., 1996) and reprinted from Pawley (2008).

calyx on the extracellular surface of human platelets that were
labeled with colloidal-gold markers and then visualized with high-
resolution in-lens cryo-SEM. All three cell-adhesion molecules in
the glycocalyx that were selected for detection could be detected
by using this method as seen in Fig. 2. In this case, after stabiliza-
tion by fixation followed by rinses in distilled water, the samples
were plunge-frozen into propane chilled with liquid nitrogen. After
partial freeze-drying at −85 ◦ C, the double-coating method devel-
oped by Walther et al. (1995) was used and involved cryo-coating
samples by evaporation of 2 nm TaW at 45◦ by electron-beam depo-
sition and 7–10 nm carbon at 90 ◦ C. This example demonstrates
that complex combination methods may be used to obtain opti-
mal results for specific projects. The choice of sample preparation
requires significant knowledge of the science project and technical
expertise, as so well demonstrated by several outstanding cell biol-
ogists who have used microscopy as their primary research tool and
advanced science as well as techniques. The double-coating tech-
nique has been used successfully in numerous other applications
including double-layer coating of yeast cells after high-pressure
freezing and freeze-fracture (reviewed in Erlandsen, 2008).
Cryotechniques have been used with great success to stabi-
lize biological tissue before viewing them with LVFESEM. Frozen
specimen can be viewed directly in the SEM by using a cold stage
which had become available when side-entry eucentric goniometer
stages were employed that can accept high-stability, cryotransfer
stage rods as typically used for TEM electron crystallography. Sev-
eral excellent results have been reported (Müller, 1992; Pawley
et al., 1991; Herter et al., 1991; Boyde, 2008; Walther, 2008).
Uncoated and slightly coated specimens have been viewed in this
mode. Cryo-coating has been applied successfully and revealed
comparable images as seen after freeze-fracture TEM analysis and
included stereo-imaging of frozen-hydrated mitochondria. Modifi-
cations of this technique have revealed stunning new insights into
intracellular structure as shown in images obtained by applying a
freeze-fracture, thaw-fix technique developed by Haggis (Haggis,
1987; Haggis and Pawley, 1988). In this case, vitreous water that
is sensitive to electron radiation is no longer present when speci-
mens are imaged. The technique involves fresh-freezing in propane,
freeze-fracture and thawing into fixative, critical point drying and
ion-beam sputter coating with Pt before imaging as shown in Fig. 3.
Stereo-imaging of such samples allows a better understanding of
complex structural interactions of cell components. Cryofixation
is currently perhaps the best method to preserve cells and tissue
in a relative native state. As has been proposed by Boyde (2008),
live-time stereo-imaging may be one step further to obtain a better
understanding of native cell features.
Other applications to view internal cell structure using cryo-
SEM include (i) cryo-microtomy of cryo-immobilized plant and
animal cells (Nusse and Van Aelsi, 1999; Walther and Müller, 1999)
that includes examining the surface of the tissue block rather than
sections by cryo-SEM; and (ii) use of a cryo-dual beam instrument
that incorporates both focusing electrons (SEM) and focusing ion
beam (FIB) columns which has been applied by Mulders (2003)
to biological samples including yeast, bacteria, and gut epithelial
cells.
As emphasized before, adequate sample preparation is critical
and several methods may be employed for comparison studies,
Fig. 12. Stereoimage of the nuclear envelope (NE) isolated from an oocyte of the
newt Notophthalamus viridescens. The nucleus was dissected into low-salt buffer
(LSB), transferred onto a cover-glass chip, extracted with 0.1% Triton X-100 in LSB,
opened with fine glass needles, fixed in 2% glutaraldehyde and 0.2% tannic acid
in LSB, postfixed in 0.1 aqueous osmium tetroxide, dehydrated with ethanol, and
critical point dried. The cytoplasmic surface of the NE is seen on the lower right,
closely packed with nuclear pore complexes (NPCs). In the upper half, we look down
on a structure consisting of an annulus with eight cylindrical particles. A central
particle is visible in most NPCs. In the lower half, we look down on the intranuclear
surface of the NE. The fishtraps, representing the intranuclear segments of the NPCs,
are clearly visible. Between the fishtraps, are scattered remnants of the lamin fibers,
disupted during swelling of the NE in LSB. Bar = 200 nm.
Reprinted with permission from Ris and Malecki (1993).
182 H. Schatten / Micron 42 (2011) 175–185

Fig. 13. (a) Stereoimage of a tangential section (250 nm thick) through an isolated NE of a Xenopus laevis oocyte, after Epon extraction. This image shows the cytoplasmic
surface with many NPCs. The NPCs have the same appearance as in whole mounts of the NE (Fig. 1). Bar = 200 nm. (b) Stereo image of a tangential section through an isolated
NE from a X. laevis oocyte. The intranuclear surface is exposed here. Several polar views of the fishtraps are visible (arrows), resembling the fishtraps seen in whole mounts.
Of special interest are the side views of fishtraps (black arrowheads), which can be obtained only with the method described in this article. Bar = 200 nm.
Reprinted with permission from Ris and Malecki (1993).

taking into consideration that some biological specimen are more
fragile than others. The very different results obtained through cor-
rect versus inadequate specimen preparation are demonstrated in
the example shown in Fig. 4 displaying cryo-TEM of polyheads of
bacteriophage T4. Detail of polyhead polygonal subunit structure
is only seen after cryo-immobilization and partial freeze-drying
while no detail is seen after conventional fixation and critical point
drying. Controlled freeze-drying coupled with high-resolution
cryo-coating of heavy metals and cryo-FESEM has demonstrated
that a resolution in the range of 2–3 nm is possible in these cases
(Chen et al., 1995; Centonze and Chen, 1995; Chen et al., 1997;
Hermann and Müller, 1991; Wepf, 1994).
LVFESEM has been used for a variety of investigations resulting
in new insights that had not been possible previously. Fig. 5 is an
example taken from Walther (2008) of high-pressure frozen and
cryofractured pancreas tissue images with SEM in the BSE mode.
Seen here are different fracture faces of the ER membranes show-
ing the smooth extraplasmic fracture face (EF) facing the lumen
and rough protoplasmic fracture face (PF) facing the cytosol. Aden-
ovirus particles imaged with high-resolution cryo-SEM of samples
imaged with secondary electrons are shown in Fig. 6. Fig. 7 dis-
plays freeze-dried and tungsten-coated actin filaments with actin
subunits clearly visible. Further examples and detailed methods
using high-resolution cryoscanning electron microscopy of biolog-
ical samples are described in Walther (2008).
Recently, cryo-LVFESEM has been used to study nuclear
envelope alterations in Herpes Simplex Virus 1-infected cells
(Wild et al., 2009) which allowed remarkable three-dimensional
insights into virus-induced nuclear pore alterations. The inner
and outer parts of the nuclear membrane could be analyzed
clearly in infected and mock-infected (control) cells as seen in
Fig. 8.
4. Specific sample preparations for specific structural and
macromolecular analysis
This section highlights specific applications that have been
worked out by individual investigators to address specific research
questions yielding results that could only have been obtained
with LVFESEM and no other microscopy technologies. It further
highlights the importance of understanding the specimen itself,
the science, and the capabilities/limitations of instrument and
methods. The projects presented here have been possible as a
result of the investigators’ unique expertise. Three main examples
are highlighted which are (1) chromosome structure, (2) isolated
mitotic spindles, centrosoms, and nuclear envelopes, and (3) de-
 H. Schatten / Micron 42 (2011) 175–185 183

Fig. 14. (a) Stereo image of a cross-section (250 nm thick) through an isolated NE from an oocyte of Xenopus laevis. A fishtrap in profile is visible at the arrow. Bar = 200 nm.
(b) Stereo image of a cross-section (250 nm thick) through an isolated NE from a X. laevis oocyte. A side view of a fishtrap is seen at the arrows. The tips of the arrows point to
the small ring at the top of the fishtrap. A cable-like structure is attached to this ring. The nature of this ring is better seen in whole mounts. Bar = 200 nm. (c) Whole mount
of an isolated oocyte nucleus from X. laevis with the intranuclear surface exposed. The nucleus was isolated in LSB, attached to a glass chip, extracted with 0.1% Triton X-100
and 1% glutaraldehyde in LSB, postfixed with 1% aqueous osmium tetroxide, and critical point dried. The tops of adjacent fishtraps are connected to each other through a
complex cable system. Bar = 200 nm.
Reprinted with permission from Ris and Malecki (1993).

embedding of Epon-embedded thick sections featuring the nuclear
pore complex (NPC).
4.1. Chromosome structure
The structural details of chromosomes has been of interest for
over 120 years (Strasburger, 1888; Boveri, 1902; Sutton, 1902)
and excellent results have been reported for chromatin structural
studies with the 10 nm elementary fiber (Thoma et al., 1979), the
30 nm solenoid (Finch and Klug, 1976) and the nucleosome (Arents
et al., 1991). However, three-dimensional information was diffi-
cult to obtain with TEM although it has been successfully applied
by Ris and Witt (1981) using stereo-imaging with high-voltage
TEM. High-resolution SEM provided a breakthrough in analyzing
three-dimensional chromatin organization (reviewed in Wanner
and Schroeder-Reiter, 2008) and has also made it possible to dis-
184 H. Schatten / Micron 42 (2011) 175–185
tinguish DNA and proteins which had not been possible with TEM
because of the TEM staining requirements including osmium and
uranyl acetate. By using experimental manipulations and various
labeling techniques details in chromosome substructure could be
visualized and analyzed with FESEM (reviewed in Wanner and
Schroeder-Reiter, 2008). High-resolution SEM provided for the first
time details that had not been visualized previously, including an
unusual nuclear organizing structure (NOR). Two examples of this
work are shown in Figs. 9 and 10 taken from Wanner and Schroeder-
Reiter (2008).
4.2. Isolated mitotic spindles, centrosomes, and nuclear envelopes
As for chromosomes described above, isolated cell components
with a complex surface topography must be coated with metals
without obscuring structural details, presenting a challenge for
many samples including the isolated mitotic apparatus (MA) and
centrosomes. As for chromosomes, the isolated MA had previously
been visualized with high-voltage TEM or negative staining but
details in surface structure could not be seen with these techniques.
By using LVFESEM, it was possible to visualize microtubules ema-
nating from a central core, decorated with components that are
tightly attached to microtubules. Vesicles were co-isolated along
microtubules (Fig. 11). An isolated nuclear envelope is shown in
Fig. 12.
4.3. De-embedding of Epon-embedded thick sections
De-embedding of thick-sectioned cells and tissue offers unique
insights into internal cell structures. The methods for this very dif-
ferent specimen preparation approach are described in more detail
by Schatten (2008) reviewing various applications including the
nuclear pore complex (NPC), insect flight muscle (Ris and Malecki,
1993), Ascaris (Sepsenwol, 2006) and internal structures of the
apicomplexan parasite Toxoplasma gondii (Schatten and Ris, 2002,
2004; Schatten et al., 2003). In the present review, one representa-
tive application is chosen featuring the NPC that had originally been
elaborated with great care by the late Hans Ris (Ris and Malecki,
1993).
The study of the nuclear pore complex (NPC) had been facilitated
greatly by using HRLVFESEM of thick-sectioned material coupled
with stereo-imaging. Previous studies had used negative staining
and thin-section TEM which only revealed part of this complex
structural component of the nuclear envelope. LVFESEM analysis
showed for the first time intranuclear components that had not
been possible with any other method applied so far. The images
showed details of the cytoplasmic part and the intranuclear part in
three dimensions revealing that the cytoplasmic ring consisted of
short rods twice as long in height than in width. The nuclear pore
complex structure was termed fishtrap by Hans Ris who showed by
using LVFESEM that the inside of the NPC consists of a ring 120 nm
in diameter with eight thin filaments projecting from it into the
nuclear space. The ends of these filaments are attached to a smaller
ring of about 60 nm in width. The comparison with TEM showed
that TEM was only partially able to capture these delicate struc-
tural features. Figs. 13 and 14 are representative images of nuclear
pore details as viewed with HRFESEM after de-embedding of thick
sections.
5. Future perspectives
As emphasized in this review, the preservation of native cellular
events afforded by controlled chemical fixation and/or cryo-
preservation of delicate cellular structures is among the aspects
important for future developments in HRLVFESEM. The devel-
opment of high-resolution variable pressure instruments are in
progress as well as other instrument developments related to SEM
to allow imaging of cellular structures that are closer to live-cell
conditions than currently possible and require modified versions of
SEM. So far, excellent combination methods have been employed
to assess live conditions observed with light microscopy followed
by analysis with electron microscopy (Knott et al., 2009). Stud-
ies by these authors and others identified structures of interest
with live confocal microscopy followed by analysis with focused
ion beam/scanning electron microscopy (FIB/SEM) and serial block
face/scanning electron microscopy (SBF/SEM). Serial section SEM
of adult brain tissue using focused ion beam milling allowed visu-
alization of the ultrastructure of a time-limited biological event
within the context of a whole organism (Knott et al., 2008). These
studies and others offer new directions into an area of future appli-
cations aimed at studying live-like events at high resolution. It can
be expected that new developments in SEM will play a major role
in such studies.
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