Sequential Replicas for In Vivo Imaging of Growing Organ Surfaces - Plant Cell Morphogenesis: Methods and Protocols - page 105

4. The time for the mold setting depends on temperature: the
higher the temperature, the shorter the time, and we advise to
minimize the time of organ surface contact with the dental
polymer (we observe surface damage after leaving the polymer
on the organ overnight; also, the presence of set polymer on
the organ surface is in fact a mechanical factor). However, too
high temperature may cause organ desiccation and makes the
time window, when the dental polymer is fl uid enough to be
applied on the surface of interest, too short. Thus, the tem-
perature cannot be too high. On the other hand, a lower tem-
perature (below 16 °C) may increase the setting time to several
hours or even prevent the setting of the dental polymer.
5. Using the scaffolding is necessary especially when apices or
apical meristems of elongated shoots in the vegetative phase of
development are studied [ 10 , 14 ]. In case of some organs,
whose surface is easier accessible, this step can be much simpli-
fi ed. For example, in the case of the infl orescence shoot apex
(or apical meristem) of Arabidopsis thaliana [ 15 ], it is enough
to bend away young fl ower primordia with a sharpened end of
a toothpick—they often remain in such a changed position for
a moment long enough to apply the dental polymer. Young
leaves or stems of many species, in turn, do not require any
additional operation at all.
6. It is good to check the appearance of the mold surface imme-
diately after taking (e.g., using a stereomicroscope). Sometimes
the polymer has not reached the surface of interest or there is
an air bubble on this surface. This can be recognized already
in the mold and the next trial to obtain the mold can be made
before water is applied to the organ surface. Such repetition
should, of course, be avoided if possible.
7. We observed that growth of shoot apices [ 14 - 16 ] and young
leaves, e.g., 1-5 mm long leaves of Arabidopsis thaliana [ 13 ]
or Anagallis arvensis , apparently slows down or even ceases
after four successive replicas have been taken. The minimal
time interval that we recommend for shoot apices is 10 h [ 15 ]
and for expanding leaves, 48 h. If the interval is too long, the
recognition of the same region (individual cells and their
progeny) may be diffi cult or even impossible in consecutive
replicas because too many cell divisions take place during that
time. On the other hand if the interval is too short, differences
in the cell size between the consecutive time points may be too
small for growth computation.
8. If a surface studied is fl at, the cast can be made also from nail
polish and observed under light microscope in a drop of water
or 50 % glycerol solution. The nail polish has to be transparent
[ 13 , 17 ].