LABEL-FREE MONITORING OF CELL SIGNALLING PROCESSES THROUGH AFM-BASED FORCE MEASUREMENTS - Life at the Nanoscale: Atomic Force Microscopy of Live Cells - page 350

luorescence microscope to elucidate the cellular responses associated with
the activation of cell receptors by selective biochemical stimuli. In a typical
experiment, an AFM cantilever mounted with a tip of 50 nm in radius is
allowed to contact the cell surface with a force inferior to 250 pN. Using an
X-Y piezo stage, the tip can be precisely positioned over speciic regions of
an individual cell. In this procedure, the positioning precision is normally
limited by the resolution of the optical microscope under which the AFM-
based force measurement instrument is mounted. The highest part of the
apical region (i.e. the nucleus) is often selected to minimize variation in
the cellular response that could be associated with the varying geometry
throughout the cell surface.
Activation of pathways leading to minute morphological changes
in the cell body is detectable through AFM-based force experiments
as demonstrated using HEK-293 cell line stably transfected with the
angiotensin receptor (AT 1 -R),

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which is a model for cell responding to

angiotensin II (AngII). This receptor is well known to induce cellular

response in endothelial cells and smooth muscle cells controlling blood low
and pressure. Figure 17.2 represents typical AngII-induced cellular response
detected with the AFM cantilever, plotted as cell membrane displacement
(nm) as a function of time (minutes). The response could be alternatively
plotted in force unit (pN), using the spring constant of the cantilevers, which
are usually chosen in the 0.005-0.015 N/m range for optimum sensitivity.
Prior to stimulation, the baseline is recorded for several minutes to ensure
thermal and mechanical stability. During this period, the signal exhibits an
average height luctuation of 0.70 ± 0.07 nm, corresponding to 7.0 ± 0.7
pN, which mainly consist of inherent cantilever noise. An important feature
of these curves is the large cell membrane displacement (262 ± 52 nm,

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n

= 6 independent experiments) observed immediately after the stimulation
by AngII (100 nM), which occurs as a result of the cell developing a
positive force against the tip. Simultaneously recording of phase contrast
micrographs conirms that the change in the AFM signal originates from
minute morphological changes occurring throughout the cell body ( Fig .
17.2b ) . The comparison of the cell morphology before ( 2 minutes) and

at the maximum of the height signal (~2 minutes) shows a contraction of
the cells bodies that is related to the AT 1 receptor stimulation. The extent of
the contraction is assessed (Fig. 17.2c) by delineating the cell body before
contraction as a standard for comparison at different stages of the cell
response. For this particular cell, a contraction is observed at ~2 minutes,
which leads to an elevation of the apical regions of the cells as measured
with the AFM. This conirms that the signal detected with the AFM relects
the structural and mechanical changes in the cells. The variability in the