Growth and Development of higher plants

Cellulose microfibrils tethered by hemicelluloses are considered as the load bearing component of plant cell walls. It is the orientation of cellulose fibrils that is a key component in the determination of the mechanical properties of cell walls. These in turn determine the direction of (isotropic or anisotropic) cell expansion. Two optical approaches are in current use to study the orientation of cellulose fibrils in the primary cell walls of Arabidopsis seedlings: polarization confocal microscopy and Field Emission Scanning Electron Microscopy (FESEM).
The first approach is done on whole plant organs in vivo.  The dichroic properties of Congo red stained cell wall components is used to determine the mean or net cellulose fibril orientation of a cell wall.  Combining this way of analysis with extensometry, the net cellulose orientation has recently been linked with anisotropy of cell wall mechanical properties as well in a monocot as in a dicot.
FESEM makes use of a scanning electron microscope equipped with a field emission electron source. This type of sources produces scanning electron micrographic images at outstandingly high spatial resolution by providing small diameter beams of high brightness and low accelerating voltages. This high resolution method produces accurate images of the recently deposited cellulose microfibrils, and is thus critical for elucidating the role these elements play in cell expansion. The protocol used, involves paraformaldehyde fixation and cryo-sectioning. The section is then treated with sodium hypochorite resulting in extraction of remnants of the cytoplasm and of cell wall matrix components, critical pint dried and gold coated. This approach allows to view the innermost layer of cell walls at any position along roots, hypocotyls and leaves. Maintainance of basic anatomical characteristics of the organs allows the positioning and the orientation of the cell wall(s) visualised in the electron micrograph.
The equipment used are: a confocal microcope NIKON C1, a cryo-ultra-microtome (Sorvall MT-2) with a low temperature controller (Sorvall LTC-2), a critical point drier (Balzers CPD 010) using liquid CO2, a sputter coater (Balzers SCD 004) using a gold target, a dual beam scanning electron microcope FEI- Nova 200 NanoLab, of which the FESEM function is used.