Sequential Replicas for In Vivo Imaging of Growing Organ Surfaces - Plant Cell Morphogenesis: Methods and Protocols - page 96

Chapter 8

Sequential Replicas for In Vivo Imaging
of Growing Organ Surfaces

Dorota Kwiatkowska and Agata Burian

Abstract

Sequential replica method facilitates in vivo imaging of plant surface and provides data suffi cient for
detailed computation of geometry and growth. It enables obtaining a series of high-resolution images
visualizing details of the examined surface. Series of molds, made in dental polymer, representing the
examined surface are used to obtain casts in epoxy resin, which are in turn observed by scanning electron
microscopy, while the structure itself remains intact. Images obtained from casts can be further used for
data extraction, comprising 3D reconstruction and computation of local geometry and cell growth param-
eters. The sequential replica method is a universal method and can be applied to image complex shapes of
a range of structures, like meristems, fl owers, stems, leaves, or various types of trichomes. Different plant
species growing in various conditions can be studied.

Key words In vivo imaging, Organ geometry, Cell growth, Epidermis, Shoot apical meristem

1 Introduction

Plant morphogenesis comprises maintenance and changes of organ
shape (geometry) that can be realized by various growth patterns
[ 1 , 2 ]. In order to understand how morphogenesis is regulated,
one usually needs to perform detailed quantifi cation of geometry
and growth of the developing organ. This quantifi cation is often a
challenge in technical and computational terms because, in most
organs, geometry and growth patterns are complex and changing
in time. If changes of a complex organ shape are to be quantifi ed,
3D reconstruction of the organ surface is required. Moreover, if
growth pattern is changing in time, as in majority of developing
organs, sequential imaging in vivo is required for growth quantifi -
cation. Noteworthy, there are only few cases when temporal changes
of growth do not have to be accounted for; accordingly one-time
examination of the organ is enough for growth quantifi cation.
These exceptions are root apices [ 3 ] or shoot apical meristems with
relatively small primordia [ 4 ] considered within a short time frame.