Imaging Plant Nuclei and Membrane-Associated Cytoskeleton by Field Emission Scanning Electron Microscopy - Plant Cell Morphogenesis: Methods and Protocols - page 167

most important to choose an appropriate and gentle fi xation tech-
nique to preserve the cellular content in as close to in vivo state as
possible. A variety of fi xation methods can be tested for the desired
structural preservation [ 1 - 3 ]. Because fi eld emission in-lens SEM
(feiSEM) achieves better than 0.5 nm resolution, it is a powerful
tool to provide structural information complementary to light
microscopy. Labelling of proteins and other macromolecules with
antibodies conjugated to gold particles helps with interpretation of
the data.
For feiSEM the surface of interest must be exposed. If the cell
wall of plant culture cells is of interest, simply wash off the medium
and replace it with a suitable fi x. If the surface is buried deep in the
cell or integrated into a tissue, it may be necessary to test a variety
of options.
Some cellular components can be uncovered by fracturing
techniques such as freeze fracture or dry fracturing [ 4 ], but these
methods usually preclude immunolabelling because the sample is
either frozen or fi xed and dried, respectively, before the labelling
can be done. For many intracellular components it may be neces-
sary to isolate them. In this way membranes can be preserved, but
morphological changes are possible. Unlike purifi cation of organ-
elles for biochemical analysis, however, it is unnecessary to aim for
highly purifi ed preparations where damage is likely to occur. We
have developed methods for isolating nuclei from plants suitable
for SEM and feiSEM and immunogold labelling [ 5 ]. The proce-
dure involves removal of the cell wall and then bursting the result-
ing protoplasts either osmotically or using mechanical pressure,
e.g., pushing through a syringe needle or wet fracturing as described
in the method of Collings et al. [ 6 ]. This technique can be similarly
adapted when other plant cell types such as leaf or root cells and
their organelles are of interest; protocols for making protoplasts
from Arabidopsis or tobacco leaf or root cells can be found in [ 7 , 8 ].
Handling protoplasts requires some experience, though. It is
important to choose the appropriate enzyme cocktail for cell wall
removal and to ensure the enzymes would not cause any unwanted
damage to the cellular content by washing off thoroughly before
bursting the protoplasts.
Prior to imaging by SEM, the procedure further involves post-
fi xation of membranes with osmium tetroxide, dehydration of the
sample, and metal coating, details of which can be found in [ 9 ]. In
the protocol presented here, we describe how to isolate and fi x
nuclei of plant culture cells for feiSEM (Fig. 1 ). The method can
be easily adapted for observing the membrane-associated cytoskel-
etal structures (also detailed in the protocol; Fig. 2 ) or nuclei of
leaf or root cells (by adapting the protoplasting procedure
accordingly).