Sequential Replicas for In Vivo Imaging of Growing Organ Surfaces - Plant Cell Morphogenesis: Methods and Protocols - page 104

operation you may decrease the magnifi cation temporarily but
you cannot use rotation with respect to the Z -axis. Images with
a scale bar pasted are preferable. Information about the exact
tilt angle, magnifi cation, and work distance is required for each
image for further analysis, i.e., 3D reconstruction, curvature,
and growth computation [ 8 ].

4 Notes

1. The surface to which the polymer is applied must be really dry.
If there is even a small amount of water or other liquid on the
examined surface, the polymer will not reproduce the surface
details (no visible cells) or may not set. According to product
information and our own experience, dental silicon polymers
used for molds do not set in the presence of sulfur compounds.
In such cases often an oily and sticky fi lm is formed on the
organ surface that cannot be removed, and thus the specimen
is lost. This can be a serious problem in case of species that
produce sulfur-containing exudates on the organ surface or
with sulfur compounds in a cell sap, since it is often unavoid-
able that some tissue in the vicinity of examined region is dam-
aged during mold taking. It may also be the reason why it is
often virtually impossible to obtain good molds from
Arabidopsis thaliana plants that have been attacked by aphids
or other pests ( see Fig. 3c ) because sulfur compounds can be
produced in reaction to pests in myrosin cells of plants from
Brassicaceae family [ 11 , 12 ].
2. The sequential replica method can be combined with in vivo
confocal laser scanning microscopy (CLSM) ( see Chapter 9 ) .
In this case plants are grown in in vitro culture on agar-
solidifi ed medium. Replica method can be combined also with
other methods. For example, the specimen can be fi xed imme-
diately after the last replica has been taken and used for
β -glucuronidase reaction, nucleus staining [ 13 ], or analysis of

inner cell wall pattern. Although it may be time-consuming, it
is possible to recognize the same cells in the replica and fi xed
material ( see also Note 11 ).
3. The composition of products is changing and the manufactur-
ing companies adjust them to dentist/patient requirements
that are not always the same as those for replica taking from
plant material. Therefore, we recommend checking the
product before usage, e.g., applying it to an easily accessible
shoot surface, obtaining a mold, and checking for epidermal
cells viability next day. The disadvantage of the new products
is in the addition of components that make them more hydro-
philic, which may possibly enhance plant cell desiccation.