LABEL-FREE MONITORING OF CELL SIGNALLING PROCESSES THROUGH AFM-BASED FORCE MEASUREMENTS - Life at the Nanoscale: Atomic Force Microscopy of Live Cells - page 353

stimulation. Such control experiments conirm that the AFM makes it
possible to detect the activation of AT 1 receptor ( Fig. 17.2 ) and its effect on
the cells mechanical homeostasis.
17.3 AFMBASED MEASUREMENT AND FLUORESCENCE
IMAGING OF CELL CONSTITUENTS
Because of its great sensitivity, the AFM can detect morphological changes
of small magnitude, which are not readily detectable with conventional
optical imaging techniques. The visualization of molecular, ionic, structural
and morphological changes, occurring within the cell simultaneously
to force signal monitoring, offers a signiicant advantage in the study of
receptor-dependent cell responses.
Using appropriate luorescent markers, it is possible to visualize the
contractile response of the cell body, actin rearrangement, organelle
movement, receptor internalization and chemical (pH, Ca 2+ ) changes
occurring within the cell. The observation of these events and their
quantiication make possible a rational interpretation of the features seen in
the AFM signal and provide independent cues conirming the contribution
of speciic signalling pathways in the morphological and mechanical changes
in the cell occurring as a result of receptor activation. In this section,
we illustrate how actin cytoskeleton visualization and the monitoring of
intracellular calcium can be used in conjunction with AFM-based force
experiments on receptor stimulated cells.
17.3.1 Contribuon of the Acn Cytoskeleton in the AFM Force
Signal
Contribution of actin cytoskeleton in the recorded AFM signal can be
assessed using HEK-293 cells expressing the AT 1 receptor and co-transfected
with GFP-actin to generate luorescence micrographs at different times
after stimulation of living cells. 23,25,26 Correspondence between the AFM
signal and confocal micrographs, showing the actin cytoskeleton at
different times before and after AngII stimulation, is presented in Fig. 17.4 .
The confocal micrograph of the basal section ( Fig. 17.4b at −2 minutes),
obtained prior to AT 1 stimulation, shows a morphologically stable cell with
numerous actin structures. 25 A transversal confocal micrograph of a cell is
presented in Fig. 17.4c and shows a relatively uniform distribution of the
GFP-actin throughout the cell body prior to the stimulation (−2 minutes).
This transversal presentation does not easily allow for the observation