For the SEM, samples are fixed in glutaraldehyde, dehydrated through a series of
solvents and dried completely either in air or bycritical point drying.This method
removes all of the water from the specimen instantly and avoids surface tension in the
drying process thereby avoiding artifacts of drying. The specimens are then mounted
onto a special metal holder orstuband coated with a thin layer of gold before viewing
in the SEM (Fig. 4.22, see also colour section). Surfaces can also be viewed in the TEM
using either negative stains or carbon replicas of air-dried specimens (Fig. 4.21).
Immuno-EM methods allow the localisation of molecules within the cellular
microenvironment for TEM and on the cell surface for SEM (Fig. 4.23). Cells are
prepared in a similar way to indirect immunofluorescence, with the exception that
rather than a fluorescent probe bound to the secondary antibody, electron dense
colloidal gold particles (10 nm) are used. Multiple labelling can be achieved using
different sizes of gold particles attached to antibodies to the proteins of interest. The
method depends upon the binding of protein A to the gold particles since protein
A binds in turn to antibody fragments. Certain resins, for example Lowicryl and LR
White, have been formulated to allow antibodies and gold particles to be attached to
ultrathin sections for immunolabelling.4.6.3 Electron tomography
New methods of fixation continue to be developed in an attempt to avoid the artifacts of
specimen preparation and to observe the specimen more closely to its living state.
Specimens are rapidly frozen in milliseconds byhigh-pressure freezing. Under these
conditions the biochemical state of the cell is more likely to be preserved. Many of these
frozen hydrated samples can be observed directly in the EM or they can be chemically(a) (b) (c) (d)Fig. 4.22Imaging surfaces using the light microscope (stereomicroscope) and the electron microscope
(scanning electron microscope). Images produced using the stereomicroscope (a) and (b) and the scanning
electron microscope (c) and (d). A stereomicroscope view of a fly (Drosophila melanogaster) on a butterfly wing
(Precis coenia) (a) zoomed in to view the head region of the red-eyed fly (b). SEM image of a similar region of
the fly’s head (c) and zoomed more to view the individual ommatidia of the eye (d). Note that the
stereomicroscope images can be viewed in real colour whereas those produced using the SEM are in greyscale.
Colour can only be added to EM images digitally (d). (Images (b), (c) and (d) kindly provided by Georg Halder,
MD Anderson Medical Centre, Houston, USA.) (See also colour plate.)132 Microscopy