Sequential Replicas for In Vivo Imaging of Growing Organ Surfaces - Plant Cell Morphogenesis: Methods and Protocols - page 102

3.3 Preparing
the Cast

1. Mix rapidly two small equal-volume drops of the two epoxy
resin components (resin and hardener) with a toothpick for
circa 5 min.
2. Fill the molds with the resin ( see Note 8 ). Use stretched end
of thin glass tubes, toothpick with sharpened ending, or a den-
tal instrument with fi ne ending to apply the resin. If air bub-
bles appear in the resin contacting the surface of interest, the
glass tubes or toothpicks can be used to remove them gently.
It is crucial to touch only the bubbles, not the polymer sur-
face, since it could damage the mold. Do not mind small bub-
bles in the cast interior (not contacting the surface of
interest)—they will not be visible in SEM. The resin mixture
cannot be used after it became apparently more viscous (it
takes usually circa 15 min) than immediately after mixing in
order to ensure proper penetration of the mold. Make also
sure that the cast is not very thin, applying additional amount
of the already setting resin mixture if necessary. Leave the
molds fi lled with resin overnight in an open petri dish.
3. Remove gently the casts from molds using forceps ( see Note 9 )
and place them in drops of freshly mixed epoxy gel on a SEM
stub. The surface of cast in contact with the epoxy gel has to
be large and provide the hold strong enough for later cast
trimming. The epoxy gel mixture cannot be used after it
became viscous (circa 5 min from mixing), so that long threads
are drawn during application. The threads may stick to the cast
surface of interest.

4. Pay attention that the surface of interests is fully uncovered
and thus accessible for the SEM observation. This can be
achieved trimming the casts. After the epoxy gel sets strongly
(it takes circa 1 h), the excess of resin or casts of some organs
that obstruct the view ( see Fig. 3a, b ) can be removed with a
razor blade or an injection needle under stereomicroscope.
This is often tricky and you may damage the surface of interest
( see Fig. 3a ) but the casts can be obtained several times from
the same mold and trimmed again ( see Note 10 ). Before SEM
examination, get rid of all epoxy resin debris using a compressed
gas duster.

1. The requirements of the available SEM machine have to be
followed, keeping in mind that the epoxy resin casts do not
require drying but need only to be sputter-coated. The vac-
uum mode of SEM operation always gives satisfactory results
( see Note 11 ). Although in principle the environment mode
could be also used, it usually gives blurred cell outlines; thus,
we recommend the vacuum mode with sputter-coated casts.
2. If the SEM images are to be used for a 3D reconstruction,
which is crucial in the case of organs of complex geometry,

3.4 Scanning
Electron Microscopy
Examination