INSTRUMENTATION AND EXPERIMENTATION - Nanoindentation with Biological Applications - page 49

and displacement of the unconstrained indenter via calibrated stage
translation, respectively.
Frame compliance can also be measured directly, e.g ., via
compression of the indenter shaft against the sample stage, 5 or indirectly.
Indirect estimation of C f can be achieved via concurrent modification of
C f and A ( h c ) to achieve the independently verified E and H of a standard
material using a probe for which the area function is established
independently, 4 or via assumption of constant (but unknown) stiffness
and hardness of an indented sample, whereby C f is the linear
extrapolation of unloading compliance d h /d P vs. the inverse contact
depth 1/ h c . Both of these indirect methods are particularly sensitive to the
accuracy (and paucity) of data obtained at low h c , and require
assumptions of material behavior and contact that are not required of
direct frame compliance determination.
Even when these signals are well calibrated, one challenge particular
to the analysis of biological materials is identification of the contact
point - the indenter displacement beyond which true mechanical contact
with the material is established and loading commences. This is due
chiefly to the high compliance of biological materials as compared to the
stiff materials for which indenters were originally designed (and thus to
the load resolution of these instruments). However, this early stage
contact can also be complicated by electrostatic and even chemical
interactions at the probe-material interface, especially if the load train
contains flexures that allow the probe to “snap” into contact with
attractive surfaces or to resist contact with repulsive surfaces. As
biomaterials are often highly charged, such interfacial complications can
be appropriately minimized via analysis in fluid cells filled with ionic
aqueous media. Of course, any swelling associated with aqueous
immersion further increases sample compliance, and thus load resolution
may then challenge accurate contact point identification. Beyond
minimizing any user-specified, initial contact loads beyond which
data acquisition commences, it is advisable to always collect load-
displacement ( P - h ) data prior to the instrument's assumed identification
of mechanical contact initiation ( P = h = 0 upon loading) and to collect
these data beyond the instrument's assumed identification of mechanical
contact loss ( P = h = 0 upon unloading). Post processing of the full P-h