Mechanical Measurements on Living Plant Cells by Micro- indentation with Cellular Force Microscopy - Plant Cell Morphogenesis: Methods and Protocols - page 139

2. Prevent sample movements in all directions ( see Subheading
2.2 ). Let the sample recover from mechanical stress infl icted
during the preparation. In the case of epidermal peels, this can
take several hours.

3. Let the sample adapt to osmotic conditions. Changes in osmo-
larity result in modifi cation of the turgor pressure and cell
deformation. For example, the shoot apex needs at least
30 min to reach a stable size after a change of osmolarity. Long
relaxation times can be caused by viscous behavior of the cell
wall or osmoregulation. Check if the sample is stable with time
lapse microscopy both before and during the experiments
(e.g., using a confocal microscope for opaque samples, or
inverted microscope for transparent ones).
4. Check that the sample was not damaged during preparation,
for example, with PI staining, and make sure the sample and the
liquid is dust-free. Dust particles on the sensor tip can disturb
the measurements, and removing them is time consuming.

3.3 Poking
in Liquid Medium

1. When using a tungsten tip, only the tip itself should be
immersed in water ( see Note 3 ). The sensor probe should not
touch the water during the measurements. As a rule of thumb,
the tungsten tip should be only half immersed, meaning there
should be around 1 mm of water above the sample. Make sure
that the liquid is not too deep by using the image from the
side digital microscope.
2. The liquid surface tension will affect each force sensor in a dif-
ferent way, depending on the tip size and roughness. The force
or stiffness threshold used for contact detection during the
measurements should be higher than values resulting from the
liquid alone. The effects of surface tension have to be mea-
sured for each tip, by monitoring the force while moving the
tip in water without coming into contact with the sample.
3. Turgor pressure has a large infl uence on the measurements.
When measuring in medium, check the solution osmolarity
regularly, since it could change due to evaporation during
long experiments.

1. Choose the grid resolution depending on the size of surface
details. Typically, it is good to have at least 5-10 points across
one cell. Too fi ne a grid should be avoided, as the measure-
ment time will increase dramatically ( see Note 4 ). Start by
scanning small areas with a coarse grid to check if the sample
is properly stabilized.
2. The choice of indentation depth/force threshold for stiffness
measurements depends on what is being measured (turgor,
cell wall elasticity, cell wall strength). A general rule would be
to avoid too shallow indentations, which result in very small

3.4 Choice
of Parameters