Molluscan Research: Techniques for collecting, handling, preparing ...

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blunt-ended tool to make a ridge or a mound against which
the specimen can rest, or can be deposited in such a fashion
that the material will form waves or wrinkles. These
sculptural elements of the mounting medium offer additional
bonding surface to the shell (and opportunities for specimen
identification; Fig. 6). However, part of the specimen will be
obscured and 90° tilts of the SEM stage show less of the
apical and basal surface, making re-mounting necessary if all
views need to be achieved with a single specimen. The
mounting with carbon tabs is somewhat flexible; after an
initial placement the orientation can be adjusted a little by
pushing the specimen gently in the desired direction.
FIGURE 6. A gold coated atlantid heteropod mounted for SEM.
Due to the keel of the atlantid shell, it can not be mounted on its
periphery. The double sided carbon tab material was shaped into a
mound with old forceps and the specimen leaned against the
material. The aperture was put horizontally by a combination of
stage rotation and tilt, and the image of the specimen was kept
horizontal using digital image rotation; note the slope of the stub
from lower left to upper right corner. For publication, the specimen
can easily be cut out from the background. SEM operated in
variable pressure mode at 30 Pa, 20 kV, 200 pA, at 10 mm working
distance. Specimen courtesy Roger Seapy. Scale bar = 1 mm. Image
by DLG.
Carbon stickers and tape are supposed to be electrical
conductors, but some brands are only conductive along the
surface, not through the cross section of the material (R.
Burns, pers. comm.). Some, such as NEM tape (Nisshin EM
Co. Ltd., Tokyo) is conductive through its cross section.
Clear, double sided office tape can also be utilised, although
it is not conductive. Other double-sided tapes (copper,
aluminium, nickel) available from SEM supply vendors offer
various adhesive properties with which one can experiment.
Glues. Non-permanent spray glue (e.g., 3M) is
possibly the most flexible mounting medium. The spray is
designed for temporary attachment and remains plastic for a
prolonged time. It is applied by spraying a thin layer of glue
onto the stub. The thickness of the layer can be varied
depending on the specimens. The glue surface is less even
than that of the carbon tabs. While it is most useful for
specimens of more than 1 mm in size, it can be used even for
larval shells. Attempts to use spray glue with 1 mm
specimens by placing them on top of glue-covered pin heads
were unsatisfactory because the surface of the glue was too
sculptured. The spray glue is stable in high vacuum (10 -4 bar)
and in the electron beam. The major advantage of the spray
glue is its flexibility, even after coating. The glue itself is
GEIGER ET AL. (2007) MOLLUSCAN RESEARCH, VOL. 27
non-conductive and charging can be more pronounced than
with carbon tabs. Pre-sputter coating the glue makes the
surface non-sticky. Letting the glue dry overnight, or putting
it under vacuum, will make the glue more viscous prior to
mounting. This can be advantageous with very fragile
specimens as they will adhere with less surface to the glue.
For large specimens, the glue can be shaped into mounds as
detailed above for carbon stickers.
Polyvinyl acetate based (e.g. Elmer’s) glue is good for
mounting radulae and opercula, but should be avoided for
shells since its acidity will quickly (within hours) corrode the
shell. It is, however, suitable for grounding CPD specimens
and can easily be drawn out to form small conductors.
Preventing charging
Stub mounted specimens undercut all around relative to
the stub (e.g., a gastropod mounted on its periphery, or a
bivalve valve mounted concave side uppermost) will not
receive conductive metal coating on the side in ‘shadow’ and
will commonly ‘charge’ under the SEM. Once mounted,
such specimens can be coated by tilting the stub at a suitable
angle or may additionally require careful painting of a ‘wire’
of conductive material between the stub and the periphery of
the specimen. This is most easily achieved by placing a small
blob of carbon paint on a narrow wedge of paper and
inserting it between the specimen and the stub, being careful
to ensure that the material does not run up onto the surface to
be viewed.
Some of us consider the use of conductive glue such as
colloidal graphite or silver paste more hazardous than worth
while, because of the difficulty in applying the material at
small scales and capillary forces which can pull the glue over
the surface to be viewed. The often tricky painting of a
conductive wire onto the shell can be avoided with more
modern SEMs, low accelerating voltage (0.5–2 kV), variable
pressure operation, reduced probe current/spot size and using
frame integration as opposed to line integration as noise
reduction technique for imaging. Specimens may also be
sputtered multiple times for short periods of time at different
angles to coat the under surface, or a sputter coater with a
slanted and rotating specimen holder can be employed (e.g.,
Cressington 108SE with rotary/planetary/tilting sample
stage; Quorum Technologies SC7640 with RotaCota stage
RC7606). The thickness of the metal coating is 1–10 nm and
even excessive coating will not interfere with the detail in
normal shell and radular work.
SEM parameters
Most SEMs allow a multitude of imaging techniques,
with modern designs adding additional features. We
encourage the users to explore the parameter space to obtain
the best images possible. Figure 7 shows some options
applied to a rather difficult specimen. Charging effects are
accentuated, because the specimen is partly corroded, which
by itself usually makes charging worse. Additionally,
because the specimen is corroded, it is also more fragile and,
consequently, could not be thoroughly cleaned without
risking breakage. The remaining dirt also increases charging