Mechanical Measurements on Living Plant Cells by Micro- indentation with Cellular Force Microscopy - Plant Cell Morphogenesis: Methods and Protocols - page 138

high-concentration (2 % or more) low-melting-point agarose
(Sigma-Aldrich , http://www.sigmaaldrich.com ); make sure that
the sample surface is not below the agarose surface. Slides coated
with a tissue section adhesive (Biobond, BBInternational, http://
www.buybbi.com ) improve adhesion of single cells, such as tobacco
culture cells, on the substrate. Cell viability can be checked with
propidium iodide (0.1 %) staining (Sigma-Aldrich , http://www.
sigmaaldrich.com ) or, if the cells are large and transparent enough,
by monitoring cytoplasmic streaming visually.

3 Methods

In this section we describe how to produce stiffness maps on sam-
ples immersed in liquid with a CFM setup mounted on an inverted
microscope. Other applications, such as cell puncture or samples
that require a horizontal setup to avoid gravitropism, are men-
tioned in the notes.

3.1 Setup
Preparation

It is crucial to make sure that the setup and sensor are working cor-
rectly before each experiment. A good practice is to position the
robot, mount the sensor, arrange the lighting, and make all the
necessary connections to the computer, and then let the sensor
stiffness calibration run while preparing the biological samples:

1. Let the entire system warm up in order to stabilize the tem-
perature and minimize thermal expansion. Leave doors and
windows closed; turn on all lights and equipment that will be
used during the experiment several hours before use.
2. Mount the sensor ( see Note 1 for choice of sensor and tip size)
and make sure that there is as little play as possible in the sen-
sor arm (screws for the sensor are tight).
3. Perform multiple stiffness measurements on a hard surface
(clean glass slide) to check the sensor stiffness. Use a relatively
large grid (e.g., 10 × 10 points with lateral step size 10

m).
The reconstructed surface of the glass should be fl at and
(nearly) aligned with the [ x,y ] plane of the actuator. Sensor
stiffness should not vary more than a few percent between the
grid points, and the force-indentation curves should not
present drift in successive loading-unloading cycles. The sensor
stiffness should not deviate more than a few percent from
when it was fi rst used. A large decrease in sensor stiffness could
indicate damage, as they are very fragile ( see Note 2 ).

μ

1. Remove any obstacle that the sensor tip could collide with
during the measurements (e.g., older primordia around the
meristem, trichomes on the leaf surface). The sample surface
should be clean.

3.2 Sample
Preparation