Sequential Replicas for In Vivo Imaging of Growing Organ Surfaces - Plant Cell Morphogenesis: Methods and Protocols - page 106

9. It is worth time to realize that newly set epoxy resin casts are
rather elastic. Therefore, if you use a strong grip with the for-
ceps a nearly transparent cast may be “catapulted,” jumping
away far enough to be lost.
10. It is usually best to trim the specimen next day after fi lling the
mold with resin, because later on the resin becomes more and
more brittle and it may break during trimming forming cracks
in the cast portions of interest. Nevertheless, the molds can be
used several times to obtain the casts. If one is careful, it is only
rarely that the mold is damaged when fi lling with resin or
removing the casts. The molds can be stored in room tem-
perature in closed containers to avoid dust, although we do
not recommend to store them for longer than half a year
because slow deformation of molds cannot be excluded. This
time, however, is long enough to make fi rst casts, check them
in SEM, and repeat if necessary.
11. Cell outlines can be recognized in casts as groves in organ
surface that are formed above junctions of anticlinal walls to
the outer periclinal wall. In case of meristematic tissue, these
groves are rather shallow ( see Fig. 3d ), while in case of differ-
entiated cells like those of a young leaf epidermis ( see Fig. 3e ),
the groves are prominent. It is important to realize that not all
the anticlinal walls are apparent in replicas if cells are still divid-
ing. This is because newly formed walls are not immediately
visible—groves appear only after the new wall has shrunk a bit,
forming a grove on a surface. Therefore, in case of meriste-
matic tissues, some recently divided cells look as if the division
has not yet taken place.
12. It is crucial for the stereoscopic reconstruction of the cast sur-
face that the tilt is precisely with respect to X - (or Y- ) axis of
the image. Otherwise, the reconstruction with available soft-
ware is impossible. Better results can be obtained if single-stub
SEM stages are used (even with a few casts on it) instead of
multi-stub [ 8 ].
Acknowledgments
The sequential replica method has been developed by the late Paul
B. Green. While writing this chapter, we have used his numerous
indispensable advices that we have learned from Dr. Jacques Dumais,
the last graduate student of Paul. We would like to thank Drs. Zofi a
Czarna and Krystyna Heller (Electron Microscopy Laboratory,
Wrocław University of Agricultural Sciences, Poland) and Ewa
Teper (Laboratory of Scanning Electron Microscopy, Faculty of
Earth Sciences, University of Silesia) for their help with scanning
electron microscopy and Dr. Joanna Elsner (University of Silesia)