NANOPHYSIOLOGY OF CELLS, CHANNELS AND NUCLEAR PORES - Life at the Nanoscale: Atomic Force Microscopy of Live Cells - page 128

6.2.2 AFM Used as a Mechanical Nanosensor

The “tool of choice” for quantitatively measuring stiffness (given in N/m)
of living adherent endothelial cells is an AFM. In principle, the AFM is used
as a mechanical tool, i.e. the AFM tip is pressed against the cell so that the
membrane is indented. This distorts the AFM cantilever which serves as a soft
spring. The cantilever delection, measured by a laser beam relected from the
gold-coated cantilever surface, permits force-distance curves of single cells.
The slope of such curves is directly related to the force (expressed in Newton)
necessary to indent the cell for a given distance (expressed in metre). At least
two different slopes ( Fig. 6.9 ) can be identiied depending on the depth of
indentation. The initial rather lat slope (indentation depth: up to several
100 nanometres) relects the soft plasma membrane stiffness including the
cortical cytoskeleton (cell shell), while the late rather steep slope relects the
stiffness of the more rigid cell centre.

Figure 6.9. Indentation technique using atomic force microscopy. Indentation curve
with two different slopes.

The so-called force-distance curves can be obtained on single living
cells. Important parameters for reliable measurements are (i) how fast
(indentation velocity), (ii) how deep (indentation depth which is related to
the loading force) and (iii) how often (indentation frequency) force curves are
being obtained in a single cell. Endothelial cells tolerate such measurements
for hours when no more than 12 indentations per minute are performed,
indentation velocity is not exceeding 1 μm per second and indentation
depth is not beyond 20% of the cell height. Another important technical
improvement is the use of spheres mounted to the AFM tip. The spherical tips
(sphere diameter = 1 μm) gently interact with the cell surface, which results
in “low-noise” force curves. 15