Sequential Replicas for In Vivo Imaging of Growing Organ Surfaces - Plant Cell Morphogenesis: Methods and Protocols - page 97

Sequential replica method, developed by Paul B. Green and
collaborators [ 5 , 6 ], is one of the in vivo imaging methods that
provide data suffi cient for detailed computation of geometry and
growth. It enables obtaining a series of high-resolution images
visualizing details (superfi cial cell outlines, trichomes, etc.) of a
surface of individual plant organs. The organ surface has to be only
partially exposed for the method application. Series of molds, made
in dental polymers, representing the growing organ surface are
used to obtain casts in epoxy resin, which are in turn observed in
scanning electron microscopy (SEM), while the organ itself remains
intact. Images obtained from casts can be further used for data
extraction, 3D reconstruction, and computation of local geometry
(curvature) and cell growth parameters for which software are
available [ 7 , 8 ]. Furthermore, the replica method can be combined
with in vivo confocal laser scanning microscopy (ref. [ 9 ]; see also
Chapter 9 ) to complement growth and geometry data with distri-
bution of a reporter gene signal in the organ. Also, the specimens
can be fi xed after the last replica has been taken and used for vari-
ous histological procedures. The replica method is a universal
method and can be applied to image complex shapes of a range of
structures (meristems, fl owers, stems, leaves, or various types of
trichomes) of different plant species ( see Fig. 1 ), growing in various
conditions (outdoors, indoors, in in vitro culture, etc.).

2 Materials

2.1 Plant Material
and Growth Conditions

Sequential replicas can be obtained from virtually every organ sur-
face of almost every plant species as long as this surface can be kept
in a dry state for the time of mold taking (but see Note 1 ) and it is
not in contact with any other organ or a solid object that cannot be
temporarily moved away. Plants growing in a wide scope of condi-
tions can be used, including potted plants, plants growing out-
doors, or on solid media in in vitro culture ( see Note 2 ). The main
limitation is imposed by the conditions necessary during the time
of mold taking itself.

2.2 Exposing
the Organ Surface

1. Scaffolding: small disposable polystyrene petri dishes, dispos-
able syringes (dish and syringe size depends on the plant size),
thin bamboo sticks, waterproof tape, and waterproof glue.
2. Ultrafi ne threads used in ophthalmologic surgery. We use
ultrafi ne monofi lament nylon threads (manufactured by
Ethicon Corp., Somerville, USA).
3. Epoxy gel (not a regular resin!). After setting, the gel is not
brittle and has lower stiffness than a regular resin. We use
Devcon high strength 5 min epoxy gel. Since you will need only
small equal-volume amounts of the two epoxy gel components