C

33
Field Emission Scanning Electron
Microscopy and Visualization of
the Cell Interior
Terence Allen, Sandra Rutherford, Steve Murray, Siegfried Reipert, and Martin Goldberg

can also be obtained readily and displayed simultaneously alongside the secondary electron image,
producing complementary information from the
transmitted beam/specimen interactions. The use of
low accelerating voltages in FESEM has also been
shown to be of advantage, reducing charging and
penetration of the electron beam, but maintaining a
high-resolution information content. High-pressure
freezing, freeze substitution, and examination of cryohydrated specimens may all be used for FESEM
(Muller and Hermann, 1990; Walther, 2003) but can be
considered specialized and are not covered in this
article, although we do describe the use of cryoultramicrotomy and cryoabrasion as techniques to access
internal surfaces within the cell prior to conventional
imaging by FESEM. Basically, we deal with techniques
that rely on chemical preservation, followed by dehydration, critical point drying, and coating. Conventional SEM coating (up to 20nm thickness) with
sputtered gold completely obscures fine surface detail
in HRSEM and must be replaced by high-resolution
coating. We routinely coat with a 1- to 2-nm film of
chromium or tungsten, which has a grain size of 0.3 to
0.5 nm (Apkarian et al., 1990).

I. I N T R O D U C T I O N
Resolution in scanning electron microscopy (SEM)
has improved dramatically in recent years so that for
the majority of biological material, no significant differences exist in resolution between SEM and conventional transmission electron microscopy (TEM). High
brightness sources (field emission) and novel final lens
configurations have resulted in instrument resolutions of 0.5 to l nm, allowing direct, in situ, threedimensional visualization of surface detail at molecular
resolution. As all this technology relies on field emission sources of the electron beam, either by "cold"
field emission or thermally assisted "Schottky" field
emission, we refer to it as FESEM.
Surface imaging allows bulk samples to be examined without limitation of specimen thickness. Visualization of intracellular surfaces requires some means
of access, such as isolation of cell fractions or macromolecules, or in situ, via fracture, or sectioning
techniques. Cell-free systems, e.g., in vitro nuclear
formation, allow biological interfaces such as developing nuclear envelopes to be imaged directly (Goldberg et al., 1992). True three-dimensional (3D) surface
visualization can be achieved by tilting the specimen
to make stereo pairs, and accurate surface measurements can be made from computerized 3D reconstructions. The surfaces can be characterized further by
immunogold labeling, which can be unequivocally
localized by the strong backscatter signal of the gold
probes. For specimens that are thin enough to allow
electron penetration, a scanning TEM (STEM) image
Cell Biology

II. MATERIALS A N D
INSTRUMENTATION
1. Glutaraldehyde (Agar Scientific)
2. Tannic acid (TAAB Laboratories)
3. TCH (Cat. No. T-2137. Sigma)

325

Copyright 2006, Elsevier Science (USA).

All rights reserved.

326

SCANNING PROBE AND SCANNING ELECTRON MICROSCOPY

4. Osmium textroxide (Agar Scientific)

5. Uranyl acetate (Agar Scientific)
6. Molecular sieve (Merck Ltd.)
7. Arklone (trichlorotrifluoroethane) Taab Labs UK
8. HEPES (Sigma)
9. PIPES (Sigma)
10. Phosphate-buffered saline (PBS)
11. Tris-HC1
12. EDTA (Sigma)
13. Phenylmethylsulfonyl fluoride (PMSF, Sigma)
14. Percoll (Sigma)
15. Sorensen's phosphate buffer
16. Poly-L-lysine HBR, MW 150,000-300,000 (Cat.
No. P-1399 Sigma)
17. Glass coverslips, 5-7mm diameter
18. Silicon chips, 5 x 5 mm 2 (Agar Scientific Ltd.)
19. Carbon-coated support grids
20. Fine forceps for handling
21. Microcentrifuge to spin suspended material
onto coverslips
22. Microcentrifuge tubes, 1.5ml, half-filled with
polymerized EM resin, ideal for supporting coverslips,
chips, and grids during specimen deposition by
centrifugation
23 High-resolution scanning EM. Conventional
"pinhole" final lens instruments with field emission
sources will allow subcellular imaging, as will conventional transmission instruments with scanning
attachments. To date, the highest resolution achieved
has been in field emission instruments with the facility to position the specimen in, or very close to,
the final lens. Recent technology has significantly
improved the resolution of field emission instruments
at lower accelerating voltages (1 kV), from around 4 to
1.5 nm, facilitating imaging without the need for metal
coating. The microscope should also be equipped with
a suitable high-resolution backscatter detector for
immunogold labeling. The main suppliers for these
instruments are Hitachi, Jeol, Philips, and Leo.
24. Critical-point drier, with high-purity CO2
(<5 ppm water). Liquid CO2 should be passed through
a filter to remove water (Tousimis Research Corp.,
filter 8782).
25. Coating units. Oxygen, hydrocarbons, and
water vapor all adversely affect the grain size of
chromium or tantalum deposited by sputter coating
(Apkarian et al., 1990). We use an Edwards Auto 306
12-in. coating unit with cryopump, magnetron head,
and suitable power source (Edwards High Vacuum
International). Similar configurations using Denton
HiVac and Balzers equipment with cold-trapped turbopumping have also been successful. Any system
should use high-purity argon and have a shutter and
a specimen table that tilts and rotates. Film thickness

monitoring of coating deposition is an advantage.

Several "benchtop" systems are currently on the
market, many untested by the highest resolution. In
our own very recent experience, the provision of a suitably performing coating system has often proved to be
a limitation of the perceived performance of a newly
acquired microscope, and suitable resources for a highresolution coating system should be incorporated in
any application for a field emission instrument. If
possible, get in touch with an established group and
get advice before committing to any particular coating
system.

Pitfalls
It is crucial to visit manufacturers' demonstrations
with the material that will be investigated to ascertain
that suitable performance can be assured from the
chosen equipment.

III. PROCEDURES
A. Exposing Surfaces within the Cell
1. Subcellular Fractionation

Organelles and macromolecules can be isolated by

standard procedures, possibly requiring subsequent
modifications in the light of HRSEM visualization,
which are beyond the scope of this article. Basically,
the specimens must be undamaged by osmotic shock,
proteolysis, or unsuitable isolation buffers. They must
also be clean. The surface of organelles should, for
instance, be free of attached cytoskeletal remnants or
cytoplasmic contamination. Where the specimens are
available as purified macromolecules or viruses, they
may be deposited on carbon-coated TEM grids in the
conventional manner and viewed by HRSEM. In this
situation, TEM negative staining will usually be
replaced by fixation for SEM and air drying replaced
by critical-point drying followed by chromium
coating. If a STEM detector is available, the virus/
macromolecule can be recognized as a transmitted
"reference image" after this protocol and compared
directly with the secondary electron (SEM) image. The
thin coating of chromium applied for the secondary
electron imaging does not interfere with the STEM
imaging.

Adhering Sample to Support

Many cell components naturally adhere to glass
coverslips, silicon chips, or carbon support film on
grids. Glass coverslips may be a useful initial prepar-

FESEM
ative substratum, as they can be checked in the phasecontrast microscope for the density and distribution of
specimens and for the progression of various protocols
such as detergent extraction of cytoplasm. Once the
isolation protocol is established, coverslips should be
replaced with silicon chips as specimen substrates, as
silicon is a conductive substratum in contrast to glass,
an insulator, which can generate problems with charging in the SEM. Tissue culture cells will grow in identical fashion on silicon as they do on glass or plastic,
and isolated cytosol or organelles will also adhere naturally to silicon in the same w a y as they do to glass. If
samples are fixed in suspension it m a y be necessary to
coat the support with poly-L-lysine to facilitate adherence. Different samples may require slight modification, but the basic technique is as follows (Fig. 1).

Solution
Poly-L-lysine: Make a fresh 1 - m g / m l solution of
poly-L-lysine in sterile distilled water; use within 24h.

Steps
1. Mark the surface of chip or coverslip with identification n u m b e r using a d i a m o n d marker.

327

2. Place a 50-~tl drop of poly-L-lysine on coverslip

or chip; allow to stand for at least 60min in a moist
chamber to avoid drying. Rinse in sterile distilled
water. The surface will retain its adhesive properties
for up to 2 weeks in a refrigerator.
3. Place a 50-~1 drop of s u s p e n d e d material (fixed
and rinsed) on coverslip/chip. Allow to settle and
attach at 4~ and unit gravity in a moist chamber (1 h
to overnight). Alternatively, spin directly (see isolation
of nuclei) onto silicon chip.
4. Allow bulk of drop to run off, put chip/coverslip
back in fixative, and continue as for fresh tissue.
Unfixed living samples (e.g., whole cells) m a y be distorted by poly-L-lysine. To spin d o w n materials from
suspension, use minicentrifuge tubes half-filled with
polymerized EM resin to support the coverslip/silicon
chip/grid.

2. "In Situ" Exposure of Intracellular Surfaces

Dry Fracture
This is a simple but extremely effective w a y of
exposing internal surfaces in both tissues and cells.
After fixation, dehydration, and critical-point drying,

FIGURE 1 Xenopusnuclear assembly egg extract spun onto a silicon chip, fixed, frozen, and sandpapered
while frozen showing a section through the edge of a nucleus where the two membranes of the nuclear envelope can be seen, as well as the chromatin and nucleoskeletal fibres on the nuclear interior. Bar: 125nm.

328

SCANNING PROBE AND SCANNING ELECTRON MICROSCOPY

merely gently press the surface of the specimen to a

square of double-sided tape mounted to a second
silicon chip and pull away without shearing, coat both
chips as normal, and examine in the SEM. The fracture
will remove material on the surface of the adhesive
and leave fractured material "in situ.'" This technique
may be enhanced by pretreatment with detergent
(0.5% Triton X-100, 2-3min for tissue culture cells),
either alone or mixed with the primary fixative (2.0%
paraformaldehyde and 0.1% glutaraldehyde), and
subsequently refixed as described (Allen et al., 1998).

Resinless Sections
These methods involve sectioning of embedded
specimens followed by exposure of internal surfaces
by removal of the supporting material. This may vary
among epoxy resins, various waxes, and even ice.
Resins that require corrosive solvents for removal will
tend to be prone to surface etching. A mixture of 50%
propylene oxide and 50% sodium methoxide (dissolve
2g NaOH pellets in 100ml absolute methanol) will
remove most resins.

3. Cryo Methods to Expose Internal

Surfaces for FESEM
Surface imaging by FESEM may be achieved by isolating the cellular component of interest, such as mitochondria, but this approach does not allow access to
the interior structure of such an organelle (see later).
One way to expose such surfaces is to freeze the cells
or tissue and cut cryo sections, which themselves can
be viewed in the SEM, to "cryoplane" and expose the
whole blockface in the SEM, or to "cryoabrade" the
surface of the frozen sample and expose surface features in a different way. Samples are then thawed and
processed for FESEM as normal. This gives a crosssectional view but with much greater depth of information than in a resin-embedded thin section viewed
in the TEM because the sections can be very thick,
they are resin free, and there is a greater depth of focus.
Information can also be gathered quite simply in 3D
simply by taking stereo pairs, which also allows computerized 3D reconstruction and measurement of the
surface. This method is also compatible with immunogold labelling (Fig. 2).
Cryomicrotomy is an adaptation of the widely
used "Tokyasu" technique (Tokyasu, 1986, 89) for
immuno-TEM.

F I G U R E 2 Low-power image of cryo-planed block face of a mitochondrial pellet where the internal cristae structure has been
exposed. Scale bar: 667 nm.

Liquid nitrogen
Cryo ulramicrotome (e.g., Leica Ultracut R with FCS
cryo attachment)

Steps
1. Fix samples (e.g., pellet of organelles) with 4%
paraformaldehyde in PBS at room temperature for
30 rain.
2. If possible, trim to a cube of about I mm 3.
3. Transfer to 2M sucrose in PBS overnight at 4~
4. Mount sample on cryo stub for ultramicrotome
and wick off excess sucrose.
5. Plunge into liquid nitrogen.
6. Mount into a cryo ultramicrotome, which has been
precooled to-100~
7. Cut sections of 100-300 nm thickness.
8. As in the Tokyasu technique, pick up sections off
the knife on a drop of sucrose suspended from a
loop, where they thaw, and then touch the sucrose
drop to a silicon chip where the sections adhere.
9. Place chip in PBS to wash off sucrose.
10. This can then be immunogold labeled, refixed, and
processed for FESEM.

Pitfalls

Materials

These problems are associated with the Tokuyasu

technique.

Silicon chips
4% paraformaldehyde in PBS
2M sucrose in PBS
Cryo stubs

1. Poor infiltration of sample with sucrose leading to

crumbly blocks.
2. Curling of sections.
3. Static.

FESEM
4. Frosting.
5. Small sample size.
When processing the sample remaining on the pin
for SEM, the specimen always detaches from the pin.
This leaves a very small specimen that is easily lost
during the dehydration and CPD steps. An additional
problem is the attachment of the sample to a silicone
chip after CPD. The sample is very fragile and it is not
always easy to identify the "planed" side. Adhere the
sample to the chip using a thin smear of silver dag. It
is possible for the sample to flip over during this
process. Also, because of the irregular shape of the
sample, ensuring good contact and therefore a good
earth path from specimen to chip can be tricky. It is
easy to submerge the specimen in too much dag.

Cryo Abrasion
1. Centrifuge organelles onto a silicon chip.
2. Fix (e.g., 4% paraformaldehyde in PBS, room temperature for 30rain).
3. Transfer to 2M sucrose in PBS for 2h to overnight
at 4~
4. Remove from sucrose and place on filter paper to
dry the back of the chip.
5. Wick off most of the sucrose from the sample,
leaving as thin a film as possible without drying.
6. Plunge into liquid nitrogen.
7. The sucrose step can be avoided if the sample can be
frozen ultra-rapidly by plunging into liquid propane
or ethane. The chip can then be held under liquid
nitrogen or on a liquid nitrogen-cooled platform
(such as the Leica EM CPC) while it is abraded with
fine sandpaper (400-600 grade wet and dry abrasive
as per automobile body and paintwork).
8. Thaw into fix and process for FESEM.

Specific Protocol for Mitochondrial Isolation and

Exposure of Internal Structure by FESEM
Isolate mitochondria using the differential centrifugation method (Gottlieb et al., 2003). Harvest and place
cells (2.5-5 x 108) on ice for 15rain, centrifuge at 500g
for 5 rain at 4~ wash with ice-cold PBS, and subsequently wash with ice-cold mitochondrial isolation
buffer (MIB) (200raM mannitol, 70raM sucrose, 1raM
EGTA, 10raM HEPES, 0.5 mg/rnl BSA; pH 7.4). Resuspend cells in ice-cold MIB and then homogenize in a
syringe-driven cell disruptor. Spin the lysate at 800g
for 10rain at 4~ Remove supematants and spin at
10,000g for 10rain at 4~ Add fixative (3% glutaraldehyde) to the pellets and keep samples at 4~
for l h. Remove the fixative carefully and infiltrate
the pellet with sucrose/PVP solution overnight
(Tokuysau, 1989). During this process, leave the pellet

3 29

undisturbed. Then carefully excise small ( > l m m 2)

pieces of sample from the pellets and mount onto aluminium plunge freezing pins (Leica Microsystems,
Milton Keynes). Mount the pins into a plunge freeze
unit (Leica CPC) and freeze in liquid propane at a temperature of-182~ Transfer the frozen specimen and
pin under LN2 into a cryo ultramicrotome (Leica
Ultracut S with FCS attachment). Using a diamond
trimming knife (Diatome Cryotrim 45), trim several
semithin sections (350 nm) from the sample in order to
remove surface sucrose. Cut further semithin sections
(350-400nm) from the sample using a diamond cryo
knife. Collect each section on a sucrose loop according
to the "Tokuyasu" technique (Tokuyasu, 1986). Thaw
the frozen sections onto 5-mm silicone chips and
process for SEM as follows.
1. Transfer the chips with attached sections using a
metal loop (2-3 m m diameter) and invert such that the
chip is floated, section side down, in a plastic Petri dish
(35-mm Falcon) containing double-distilled water.
2. Wash 3x over 15min in order to rinse out the
sucrose.
3. Fix by floating in 1% OsO4 in double-distilled
water for lh.
4. Wash in double-distilled water 3x for 5 min each.
5. Then dehydrate the sections and critical point
dry as described later.

Pitfalls
Aligning the cryo abrasive pad with the specimen
is very tricky and must be done with care. It is very
easy for the protruding abrasive shards of the wet and
dry paper to embed themselves into the frozen block.
Also, if the section advance is too great, the sample
block can be literally ripped from the specimen pin. A
few micrometres must be shaved off the sample face
in order to ensure that all surface sucrose has been
removed. This only leaves a few micrometres of wellfrozen vitrified sample to work with. Once again, the
sample size is very small and is easily damaged or lost
in subsequent processing and mounting steps.
Identifying the abraded face can be tricky even
under a stereomicroscope. The swirled pattern of the
specimen pin that has been embossed into the underside of the sample can look very similar to the abraded
face, leading to the specimen being mounted pin
side up.

B. Fixation
Solutions
All fixatives are ideally made up just before use
or at least the same day; both glutaraldehyde and

330

SCANNING PROBE AND SCANNING ELECTRON MICROSCOPY

glutaraldehyde-tannic acid solutions should be filtered

before use through a 0.22-~tm filter. The 1% aqueous
uranyl acetate should be stored in a brown bottle.
Osmium tetroxide is made by breaking the glass
ampoules in which the crystals are delivered, having
previously washed them free of label and adhesive
under the tap, in a fume cupboard. The ampoules plus
crystals are dropped in the correct amount of buffer
or distilled water where the osmium dissolves to give
the appropriate final concentration. (Note: Osmium is
extremely hazardous and appropriate precautions
must be observed.) Thiocarbohydrazide or tannic acid
solutions should also be made just prior to use (Allen
et al., 1988).

Pitfalls
Always use glutaraldehyde of EM-grade quality
from a high stock concentration (50%) stored in a
freezer. Low concentration stock solutions and storage
in large bottles at room temperature will reduce the
cross-linking properties of the glutaraldehyde.

Steps
1. Isolated Proteins and Nucleoproteins
1. Proteins and nucleoproteins on carbon support
films on TEM grids may be floated on top of drops
(25-50~tl) of the appropriate solutions spread on
Parafilm.
2. Place in 1% glutaraldehyde in appropriate buffer for
10 rain.
3. Wash in double-distilled water for 5 min.
4. Transfer to 1% uranyl acetate for 5 min.
5. Transfer to 100% ethanol for 1-2 min.
6. Air dry or critical point dry (see later).

2. Small and Easily Preserved Structures

Steps
1. Fix in 3% glutaraldehyde in Sorensen's phosphate
buffer for 30min.
2. Wash in Sorensen's for 5 min.
3. Postfix in 1% Os04 (in Sorensen's for 30 min).
4. Wash in double-distilled water for 5 min.
5. Dehydrate through ethanol series for 5 min each.
6. Place in Arklone for 5 min.
7. Critical point dry (see later).

3. Large and/or Fragile Structures (e.g., Whole Cells,

Organelles, Cytoskeletal Preparations, Isolated
Cells, or Nuclear Membranes)

Steps
1. Attach whole cells to specimen supports such as
silicon chips and handle by changing the solutions
in 35-ram-diameter petri dishes.

2. Fix in 2% glutaraldehyde, 0.2% tannic acid, and

0.1% HEPES, pH 7.4, for 10min.
3. Wash in double-distilled water for 5 min.
4. Postfix in 0.1% Os04 in water for 10min.
5. Wash in water for 5 min.
6. Stain with 1% aqueous uranyl acetate for 10min.
7. Dehydrate through ethanol series and Arklone and
critical point dry.

4. Isolation of Nuclei from Tissue Culture Cells

Steps
1. Take approximately 10 million tissue culture cells
(usually from suspension culture), cool to 4~ and
pellet in a swing-out centrifuge (1000g for 10rain).
2. Wash the pellet in PBS buffer and then resuspend
in 8ml ice-cold swelling/shearing buffer (50mM
Tris-HC1, pH 7.4, 5mM MgCL, 1.3mM EDTA, and
5mM phenylmethylsulfonyl fluoride added shortly
before use) in which nuclei are allowed to swell for
5 min.
3. Mechanically homogenize the cells using a
plunger-type tissue grinder (e.g., Kontes Dounce),
checking the number of strokes required for nuclear
release by phase-contrast light microscopy (usually 10-20 strokes). Precool the plunger in ice prior to
use. Alternatively, nuclei may be isolated by a
single passage through a 26-G3/8 syringe needle
(Microlance).
4. Prepare a Percoll gradient as follows: use a 13.5-ml
Beckman centrifuge tube filled with 0.86ml Percoll,
density 1.130g/ml, 2.74ml 10mM Tris-HC1 (pH 7.4),
and 0.40ml 2.5M sucrose. Add density marker beads
to monitor gradient, red beads (1.12g/ml) in Percoll
containing 0.25M sucrose, and yellow beads
(1.049 g/ml) in Percoll containing 0.25 M sucrose. Spin
for 30 min at 30,000g at 4~ with a 60 ~ angle head rotor;
red beads will form a line 5 m m from the bottom of
the tube, with the yellow beads a further 12 mm above.
5. Gently layer the homogenate on top of the gradient and spin for 10min at 30,000g, which generates
two bands from the homogenate. Damaged nuclei and
whole cells are found in the upper band 7mm above
the red beads, whereas the pure nuclear fraction is
found 0.5 mm below the red beads. Remove this fraction and wash gently in 150mM Tris-HC1 (pH 7.4) for
5 min at 4~
6. Spin the isolated nuclei onto 5-mm poly-Llysine-coated silicon slips. Spin the silicon chips in
1.5-ml Eppendorf tubes previously half-filled with polymerized resin. Overlay the Si chips with 0.5 ml of freshly
isolated nuclear suspension and spin for 5 min at 1000g.
7. Fix the whole chip (+nuclei) in 6% glutaraldehyde in 0.15M Sorensen's buffer for 20min, rinse

FESEM
gently in buffer, and postfix in 1% osmium tetroxide
in 0.15M Sorensen's buffer for l h. Dehydrate, critical point dry, and coat with 3-4nm of tantalum or
chromium.

Pitfalls
1. Low yield of nuclei. Dounce tissue homogenizers are produced with different clearance distances
between the polished tube and the pestle. Some
homogenizers are designed just to disrupt tissues as a
necessary step prior to homogenization of nonsupension cells. Make sure that the clearance distance of the
Dounce tissue homogenizer for the final release of the
nuclei is small enough to disrupt whole cells. Some
manufactures offer pestles with two different clearance
distances to allow tissue disruption and release of the
nuclei within one and the same tube. For certain cell
types, making the buffer more hypotonic can help
increase the yield of nuclei.
2. Enzymatic degradation of structures. It is good
practice to ensure cooling for every preparation step
prior to the fixation of nuclei on silicon chips. Moreover,
the homogenate should be processed without delay.
Therefore, preparation of the Percoll gradient before
homogenization is recommended. To ensure a suitable
ratio between biological material and buffer, a homogenizer with a sufficient capacity (for 8ml) should be
selected. Do not reduce the number of cells significantly
to keep this ratio in a homogenizer with lower capacity,
as this might cause problems with recognition of the
nuclear layer after gradient centrifugation.

5. Preparation of in Vitro-Assembled
Organelles for FESEM
Organelles, such as nuclei, endoplasmic reticulum
(ER), and Golgi, can be assembled in cell-free extracts.
Extracts made from frog eggs are a particularly powerful system for studying the assembly, dynamics, and
functions of these organelles. Organelles can be isolated cleanly from the extract and their surfaces can be
examined by FESEM. In vitro-assembled nuclei, as well
as ER, can be prepared for FESEM as follows.
Solutions

1. Membrane wash buffer (MWB): 250mM sucrose,

50mM KC1, 50mM HEPES-NaOH (pH 8.0), l~tg/ml
aprototin, and 1 ~tg/ml leupeptin
2. Fix buffer: 150mM sucrose, 80mM PIPES-KOH
(pH 6.8), l mM MgCL, 2% paraformaldehyde, and
0.25% glutaraldehyde

Steps
1. Prepare Xenopus egg extracts (Newmeyer and
Wilson, 1991) and incubate extract with demem-

3 31

branated Xenopus sperm chromatin to assemble nuclei.

ER and Golgi will also assemble in the same extract.
2. After the required time, remove a 4-~tl extract,
place in a 1.5-ml Eppendorf tube, and resuspend very
gently in I ml of MWB. At this stage, centrifugation of
in vitro nuclei and organelles onto 5-mm 2 silicon chips
requires a simple modification of the 1.5-ml Eppendorf
centrifuge tubes as follows. Remove lid from tube and
cut tube with a sharp knife at a level where the cut end
fits tightly into the lid, thus creating a "flat-bottomed"
tube. Snap the cut end into the lid, having placed a
silicon chip in the lid first.
3. Pipette the l m l of MWB containing the extract
into the modified tube, spin in a swing-out rotor, inside
a 10-ml centrifuge tube (with a single tissue as cushion), and spin for 10min at 4~ or room temperature
at 2000g. Some leakage of the suspension at the joint
between the cut end of the tube is not a problem at this
point.
4. Pipette off most of the buffer, break open the
tube, and remove the chip. Place the chip in 5ml
of fix buffer in a small petri dish for 10min at room
temperature.
5. Wash chip in 0.2M cacodylate (pH 7.4), place in
1% OsO4 in 0.2M cacodylate for 10min, wash twice in
distilled water, and place in 1% aqueous uranyl acetate
for 10min (at room temperature) and then dehydrate,
critical point dry, etc.

C. Critical-Point Drying
All traces of water should be removed from ethanol,
Arklone, and CO2. Let 100% ethanol and Arklone stand
over molecular sieve for more than 24h prior to use.
High-purity liquid CO2 (less than 5 p p m water) should
be used and passed through a water filter as a
precaution.

Steps
1.
2.
3.
4.
5.
6.
7.

Exchange Arklone for CO2.

Flush six times.
Leave in CO2 for 30min.
Flush six times.
Raise temperature to 40~
Release gas slowly (over about 15-20min).
Transfer to coating unit as soon as possible.

Pitfalls
Critical-point-dried samples should be transferred
immediately into the sputter coater to avoid rehydration, and coated samples are best viewed in the microscope directly. However, if it is known that the
microscope cannot be accessed, it is better to pause

332

SCANNING PROBE AND SCANNING ELECTRON MICROSCOPY

preparations after critical-point drying and store preparations under vacuum.

D. Sputter Coating
Steps

1. Pump specimen to at least 5 x 10-Tmbar.

2. Introduce high-purity argon to a pressure of
8 x 10-3mbar.
3. Start specimen rotation (60rpm).
4. Sputter at 50-100mA current (voltage 450V) and
60rpm; specimen table should be tilted at 30 ~
Presputter onto the shutter for 20-60s to remove
the chromium oxide layer from the target.
5. Open shutter and deposit 2 n m chromium as indicated by a film thickness monitor (usually 20-30s).
6. Examine in microscope as soon as possible, preferably within a day or two. Coatings are variable
according to the specimen, but a general rule is that
they deteriorate with time, usually over a few days
to about 2 weeks.
E. M i c r o s c o p y

Steps
1. A liquid nitrogen-cooled decontaminator (if
present) should always be used; this is more likely to
be fitted on an "in-lens" electron optical column configuration. Many recent field emission instruments are
of conventional "pinhole configuration," but with very
short working distances to optional decontamination
devices.
2. Spot size and apertures should be as small as
possible, consistent with a sufficient signal to visualize
high resolution of specimen at photographic collection
rates (e.g., 40-s scans).
3. An appropriate accelerating voltage must be
selected. High-resolution scanning electron microscopes usually work in the range of 1-30kV. Instrument resolution decreases with decreasing accelerating
voltage; however, at high voltage there may be problems with charging and specimen penetration, leading to a nonspecific signal from below the specimen
surface. At low voltage, penetration and charging are
reduced, but so are resolution and signal. Signal is generated almost completely from the surface at 1.0 kV so
there is no problem of a "bulk" signal from underlying
structures. In general, we use high kilovolts for thin
and conductive specimens and lower kilovolts for
bulky or less conductive specimens; however, a wide
range of kilovolts should be experimented with for
each type of sample. The more recently produced field
emission instruments have vastly improved low kilo-

volt performance, and specimens that have some inherent conductivity as a result of osmium fixation can be
viewed uncoated at low kilovolts, without compromising signal and resolution.

F. Immunogold Labeling
The basics of specimen preparation for immunogold labeling are beyond the length limits for this
article and are adequately covered elsewhere (see
article by Roos et at. for additional information). For
immunogold labeling for HRSEM, the following
points are important.
1. Size of Probe

The choice of probe size is a compromise between

sensitivity and subsequent detection. Very small gold
probes (around l nm) have minimal steric hindrance
and consequently label with maximum sensitivity.
One-nanometer gold has been visualized by backscatter imaging in HRSEM (Hermann et al., 1991), but this
is at the limits of resolution and is best increased
in diameter in situ by silver or gold enhancement to a
size at which it can be visualised more easily (around
5-10nm). We have used both 5- and 10-nm gold as a
good compromise between sensitivity and localization.
Because most modern instruments will discriminate
easily between 5- and 10-nm labeling, these can be used
together successfully for double-labeling studies.
2.

Coating

Using gold probes obviously prohibits gold coating

for SEM. In the past, gold-labelled specimens have
been coated with carbon, mainly to inhibit charging,
but carbon produces a severely limited secondary electron signal and, consequently, little topographical
information. We have found that a 1.5-nm chromium
coating provides the ideal solution, retaining the full
secondary electron-generated surface information,
without compromising the detection of gold by
backscattered electron detection (Allen and Goldberg,
1993).
In this situation, having found that "mixed"
imaging of SE and BSE signals was not satisfactory, we
have chosen to collect each signal separately (but
simultaneously) and then to superimpose the gold BSE
signal onto the secondary signal (retaining register) in
Adobe Photoshop, often altering the colour to improve
the appearance of label against the monochrome background. In modern instruments with good low kilovolt performance, uncoated or carbon-coated imaging
will generate such a strong signal from gold probes
that they are observed easily in secondary electron
imaging.

FESEM

IV. COMMENTS
Although field emission SEM has been available for
some time, it is still a relatively new technique in cell
biology. The procedures given here may need to be
modified to optimize the preservation of some structures. Probably the most difficult step is exposing
recognizable and undamaged intracellular surfaces.
Isolation of organelles offers the possibilities of further
characterization by other methods, but gives no "in
situ" information and may involve extensive biochemical protocols. Resinless sections and dry fracture give
in situ information, but only after some initial extraction of the cell. Freeze fracture, followed by frozen
hydrated coating and visualization, may alleviate
these problems but is limited by the plane of fracture,
as the structure of interest may not be exposed. It is
also technically difficult and expensive. Osmium
etching results in spectacular images of intracellular
membranes, but the uncertainty of what is removed
makes interpretation difficult. Direct visualization of
biological interfaces in cell-free systems (e.g., in vitro
nuclear formation) is a particularly promising area
(Goldberg et al., 1992, 1997). Considerable fresh structural information has also been demonstrated for
nuclear pore complexes and associated structures
(Ris, 1991; Goldberg and Allen, 1992, 1996; Kiseleva
et al., 1996).
Acknowledgments

T. D. Allen, S. Rutherford, and S. Murray are supported by CRUK and M. W. Goldberg is supported by
a Wellcome Lectureship. The mitochondrial pellets
were supplied by Dr. E Gottlieb (Beatson Institute).
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