Mechanical Measurements on Living Plant Cells by Micro- indentation with Cellular Force Microscopy - Plant Cell Morphogenesis: Methods and Protocols - page 140

forces and noise from contact. For stiffness mapping, a
maximal force of 1-40 μ N for an indentation depth of

500 nm-5 m is a good place to start. The cell stiffness is in

the order of 1-50 N/m.

μ

1. Correct the position along the vertical axis for sensor defl ec-
tion, using the stiffness values obtained while indenting a hard
surface. For any position, the sensor defl ection is equal to the
force measured divided by the sensor stiffness.
2. Detect the contact point. The value of force at the contact
point can be used as an approximation of the force offset due
to surface tension, sensor drift, light, etc.
3. The stiffness can be computed in different ways. It can be
extracted as the tangent of the force-indentation curves [ 12 ]
at a fi xed force [ 15 ] or predefi ned indentation depth. If the
shape of the nonlinear force-indentation curve is known a pri-
ori, the function can be fi tted to the data [ 5 , 7 ].
4. Data interpretation of stiffness measurements is easier and less
prone to bias performed on the “fl at” parts of the sample, i.e.,
where the surface is perpendicular to sensor axis. Both the
force measured and the indentation depth are infl uenced by
the angle under which the probe touches the surface. When
comparing stiffness values, make sure that the indentation
angle did not exceed a few degrees (e.g., 10°).
5. Stiffness values do not directly refl ect the cell wall elasticity or
the cell wall turgor pressure. A mechanical model is needed to
interpret the data to extract these quantities.

3.5 Data Analysis

4 Notes

1. The choice of rounded tip of sizes larger than the cell wall
thickness ensures that the cell wall will not be damaged during
the experiment, provided that the applied force is not too
high. For cell wall puncture, use stiffer sensors (e.g., FT-S1000)
and a tungsten tip with a small tip diameter (e.g., T4-22).
2. If possible, use the same sensor (same stiffness, same gain,
same tip shape) for the whole set of experiments, in order to
compare results between samples. This also allows to check
the sensor state by monitoring its stiffness over successive days.
3. Use a side camera to check if the sensor tip is not too much
into the water. Dirt fl oating on the water surface can also
infl uence the results if it adheres to the tip. This can be pre-
vented by checking the surface regularly with the side camera
as well.