Advances in Colloid and Interface Science 120 (2006) 57 – 67

www.elsevier.com/locate/cis

Nanomechanical measurements with AFM in the elastic limit

John R. Withers, D. Eric Aston ⁎
Department of Chemical Engineering, P.O. Box 441021, University of Idaho, Moscow, Idaho 83844-1021, United States
Available online 18 May 2006

Abstract

With increasing interest in nanoscience and nanotechnology, the fundamental underpinnings of what makes materials strong and durable are
under critical investigation. Recent findings suggest that when materials are reduced in extent to nanoscopic proportions, they exhibit enhanced
strength, specifically in the form of higher moduli than are measured on macroscopic objects of the same composition. Force-deformation
behavior of nanostructures subjected to concentrated loads, such as with atomic force microscopy (AFM), can yield detailed information and
insight about their local mechanical properties. We review and evaluate the effectiveness of deformation and indentation tests used in determining
the elastic modulus of nanobeams, nanosprings, thin films, biological samples, dendrimers, and fluid droplets. Obstacles yet remain in the
determination of absolute, quantitative modulus data at the nanoscale. In spite of basic limitations, recent developments in advanced
nanomechanical techniques will facilitate improvement in our understanding of material strength and aging from molecules and colloids to the
macroscale.
© 2006 Elsevier B.V. All rights reserved.

Keywords: Atomic force microscopy; Nanomechanics; Elastic modulus; Nanoindentation; Three-point bend test

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
2. Atomic force microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
3. Bend tests. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
3.1. Nanowires . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
3.2. Nanosprings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
4. Nanoindentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
4.1. Living cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
4.2. Dendrimers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
5. Liquid interfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
6. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

1. Introduction International trends in nanoscience funding indicate the growing

popularity of his vision and the push toward nanotechnology [4–
In his classic 1959 presentation “There's Plenty of Room at the 6]. We yet understand little of the behavior of matter at the
Bottom,” Richard Feynman speculated that we would observe a nanoscale, both in general and in specific cases, even of
multitude of new material properties in substances if we could mechanical properties that are well understood at the macroscale
arrange atoms individually the way we want them [1–3]. [7–9]. Something as simple as the bending of an elastic beam or
plate must be directly confirmed or recharacterized at the
⁎ Corresponding author. Tel.: +1 208 885 6793; fax: +1 208 885 7462. nanoscale due to the basic issue of larger surface-to-volume
E-mail address: aston@uidaho.edu (D.E. Aston). ratios of these objects, where the interfacial material of the system
0001-8686/$ - see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.cis.2006.03.002
58 J.R. Withers, D.E. Aston / Advances in Colloid and Interface Science 120 (2006) 57–67
under study can have a significant effect on properties and
behaviors other than classically defined.
Traditional optical microscopy lacks the resolution to in-
vestigate phenomena of colloidal dimensions adequately, and
electron and X-ray techniques are greatly limited either by
environmental (e.g., liquids) or material property (e.g., con-
ductivity, cross-section for energy beam interactions) restric-
tions. Today, very few electron microscopes are capable of the
true atomic resolution [10] required for fundamental studies on
intermolecular and colloidal behavior of two- or three-body
interactions, for example. But scanning electron microscopes
(SEMs) have played a crucial role in the study of mechanics
with nanowires and nanosprings, and high-resolution optical
microscopy is useful for locating these so-called one-dimen-
sional objects on test substrates. In many cases, this is feasible
because of lengths exceeding a few microns that scatter enough
light for adequate contrast. The advent of atomic force micros-
copy (AFM) [11–13] marked the beginning of significant ad-
vancement toward more routine molecular-scale imaging in
three quantified dimensions with the simultaneous measure-
ment of an additional one or more physical properties. While the
literature shows a nearly exponential increase in studies of
paired interactions and nanomaterials, our ability to construct
“from the bottom up” requires a still deeper understanding and
further advances in many disciplines for Feynman's visions to
become reality.
Researchers employ the AFM in various ways to determine
sample mechanical properties [14–17], especially the elastic, or
Young's, modulus. These generally include nanodeformation
tests, such as three-point bend tests and the more common
nanoindentation. We examine the application of these methods
in the mechanical study of nanostructures, evaluating their
utility and effectiveness in estimating elastic moduli and related
mechanics.
2. Atomic force microscopy
Atomic force microscopes are themselves nanomechanical
instruments. AFM employs a sharp, cantilever-mounted probe to
raster scan surfaces. Image resolution can be very high–scientists
have observed subatomic-scale features [18]–but depends on a
variety of factors including tip sharpness, acoustic isolation of the
instrument, sampling medium, AFM controller precision, etc. The
tip and sample positions are manipulated relative to each other
with piezoelectric or other (e.g., electromagnetic coils) actuators.
The AFM precisely controls the tip location on the sample by
managing the voltage applied to the scanners [3,12]. These are
arranged either with three independent, orthogonal piezoelectric
blocks or in a tube configuration. Piezoelectric scanner perfor-
mance can be limited due to nonlinearities in the scanner, material
creep, noise and drift in the high voltage supply, or thermal drift of
the AFM apparatus itself [3,12,19].
A variety of attractive and repulsive forces act between the
tip and sample [3,12,20], such as van der Waals, electrostatic,
and capillary forces. To some extent, such forces can be con-
trolled by altering the sampling medium; for example, sampling
under water can eliminate the effect of capillary forces. Typical-
ly, a diode laser reflects off the back of the cantilever onto a
quadrant photodetector, which senses cantilever bending and
twisting. If the cantilever spring constant is known, the can-
tilever deflections may be converted to quantitative force data.
Cantilever calibration is not a trivial procedure, however. Can-
tilevers are sold with typical force constants that may, in fact,
vary by an order of magnitude from reported average values.
Alternately, the values can be calculated from the cantilever's
spring geometry [3,12,19,21,22]. For a uniform, rectangular
cross-section, the cantilever's spring constant is given by
kc = Ewt3/4l3, where w is the cantilever's width, l is its length,
t is its thickness, and E is the elastic modulus. This, however, is
seldom accurate enough for the required precision of quanti-
tative AFM studies. Most cantilever probes are rectangular or
triangular with a “two-beam” geometry connecting at the tip.
Many AFM cantilevers are also coated with one or more layers
of metal for reflectivity and other surface modifications.
From the deflection of the cantilever, we calculate tip-sample
force data using Hooke's law:
F ¼ kc z; ð1Þ
where F is the magnitude of the force acting between the tip and
sample, kc is the cantilever spring constant, and z is the can-
tilever deflection at its free end.
AFM may be known best for its ability to generate high-
resolution topographical images. In most imaging modes, a
feedback system senses instantaneous cantilever deflection and
adjusts scanner elements to maintain a constant interaction
between the tip and sample. The instrument records and plots
scanner adjustments as surface topography. In fact, the AFM
may operate in any of various modes, depending on the inter-
action energies or forces of interest. For example, selective
chemical functional groups may be attached to the probe,
generating force data reflecting sample composition. The image
produced will thus be a convolution map of chemical makeup,
not merely surface topography [23,24]. Also, the AFM can
record twisting movements of the cantilever, which represent
frictional forces acting between the tip and sample [25].
Force spectroscopy generates a force–distance curve for a
single location on the sample. This is a plot of the magnitude of
the force acting between tip and sample versus the position of
the scanner in the direction normal to the substrate. Force–
distance curves hold a wealth of information about the sample's
mechanical properties. Points of discontinuity, the slopes of the
approach and retract curves, as well as any observed hysteresis
all cede hints to surface behavior [12,26–29]. The difficulty
arises in interpretation and deconvolution of multiple phenom-
ena. Hysteresis, for example, is the result of adhesion, surface
deformation, and/or nonlinear performance of the instrument,
such as piezoelectric or other transducers for scanning and the
probe detection sensor, e.g., photodiode.
Another consideration is that the AFM does not directly
measure the actual tip-sample separation distance. Rather, it
controls and/or measures the vertical scanner position, the can-
tilever deflection, and the sample deformation [28]. We often
collect an array of force–distance curves at discrete sites over an
 J.R. Withers, D.E. Aston / Advances in Colloid and Interface Science 120 (2006) 57–67
area of the sample surface to determine effects of sample
heterogeneity. This enables the AFM to produce spatially re-
solved maps of both topography and other sample material
properties near the surface that are gleaned from force or energy
profiles. Commercially available tools, such as pulsed-force
mode (PFM) [30–32] and force-volume imaging [33], accom-
plish this mapping with improved automation. In these cases,
force, adhesion, and stiffness data are readily collected and
made available for offline interpretation and analysis. Other
physical interactions between the probe and sample may also be
mapped with more difficulty, such as energy loss or long-range
forces. It is primarily the stiffness data that are of interest to
nanomechanical studies.
3. Bend tests
Nanowires and nanosprings have been highly studied for
their sometimes remarkable electrical and mechanical properties
[15,34–39]. Some nanowires exhibit surprisingly high tensile
strength, possibly due to the presence of fewer mechanical defects
per unit length than in their macroscopic analog [39]. Being able
to synthesize these structures reliably and understand their
behavior will be vital to their ultimate application as future
building blocks in everything from circuits to microelectrome-
chanical or nanoelectromechanical systems (MEMS or NEMS),
environmental sensors to biomimetic implants, hydrogen pro-
duction and storage to smart composite materials, just to name the
more popular examples [4–6].
Deformation tests to determine the mechanical properties of
edge-supported films are similar to bending nanowires and are
plagued with similar difficulties of experimental setup and data
interpretation at the nanoscale. While a significant literature
base exists on bulge tests [40], the “two-dimensional” analog of
beam bending, there is, surprisingly, little in the way of true
nanomechanical investigations with AFM [41], nanoindentation
[42], or other techniques, that is, nanoscopic in both morpho-
logy and stress, e.g., nanoNewton point loads. Edge-supported
film deflections may be interpreted from expressions derived for
the classic centrally loaded plate deformations [9] or from
extended models for membranes and shape memory materials
[42]. Yang [41] also described research in which polymer films
Fig. 1. Schematic representation of beam support models: (a) simple and (b) clamped.
59
of varying thickness were bound to copper grids, resulting in
adhesive, edge-clamped, free-standing films over each grid
square. The films subjected to point loading with AFM were
modeled as beams but are closer to rectangular plates, even though
significant stretching of the polymeric (membrane) material may
be expected to skew the results significantly from classic behavior.
Local deformations in AFM bend tests cannot be neglected as in
some classic experiments, but a satisfactory solution for nano-
mechanics is yet to be developed. We expect to observe discrep-
ancies between actual deformations and predicted values based
only on global bending model predictions at this stage in devel-
opment for both suspended films and nanowires [17].
3.1. Nanowires
Researchers employ various deformation tests to study the
moduli of elasticity of nanowires. In general, the expressions used
to relate elastic modulus to observed deflection and applied force
come from classic texts on strength of macroscopic materials. This
approach is valid when the beams follow linear elastic theory of
isotropic materials and have high length-to-thickness ratios [8,34].
A beam can be supported in various ways resulting in mathe-
matical description via boundary conditions of the type of sup-
ported ends. The two extremes in behavior are set by having both
ends either free or built-in (that is, clamped or fixed) to resist both
torque and slip. The first is the simply supported beam (Fig. 1a)
and the latter is the clamped end configuration (Fig. 1b). Other
beam bending behavior may be described by mixed boundary
conditions, or by modifications thereof, for example, a limited-slip
or limited-torque end that might be related to adhesion and
tribology.
The general bending equation expresses beam deflection, z,
in the plane of applied stress as a function of the beam's
moment, M [7]:
d2 z
EI ¼ ¼ −M : ð2Þ
dx2
The first moment of inertia, I, depends on the beam's cross-
sectional shape and physical dimensions. (If the beam shape
changes during measurement as may be expected for nano-
mechanical studies, the in situ determination of I may be
60 J.R. Withers, D.E. Aston / Advances in Colloid and Interface Science 120 (2006) 57–67

intractable for nanowires–or even unsolvable under AFM test segment of constant compliance. Young's modulus can then be
designs–and will at worst need to be considered a second computed from Eq. (7) with kw and the geometry of the nano-
variable parameter for interpretive analysis [17]; this will be a wire setup (from AFM and/or SEM measurements).
future topic for expansion.) The moment M depends on the We find that for beams with identical material properties and
magnitude of the concentrated point load, F, the load location physical dimensions, where the only difference is the support
measured from the origin down the long axis (i.e., the x- scheme, the midpoint deflection for the simply supported beam
coordinate), a, and the length of the beam, L. M is negative will be larger by a factor of four, as shown in Fig. 2 [7]. This
when the z-axis is defined as positive in the downward idealized scenario is of course complicated for systems where
direction, convenient for AFM bending studies. In the case of the supporting substrate contributes to the overall compliance
built-in or clamped ends, M is expressed as being measured by the AFM experiments. That is, there will be
multiple deformations convoluted into the process if the sup-
Fb Fab2 Fa2 b ports for the nanowire ends are not themselves rigid or if the
M¼ x− 3 ð L−xÞ− 3 x; x V a; ð3Þ
L L L supported portions of the nanowire itself distort during the test.
and We are currently investigating models for “springy” and sliding
supported ends that allow these additional deformations to be
Fb Fab2 Fa2 b
M¼ x−F ð x−aÞ− 3 ð L−xÞ− 3 x; xza: ð4Þ fitted; however, under these scenarios with multiple unknown
L L L parameters, the goal of quantifying nanowire elasticity with
Substitution of Eq. (3) or Eq. (4) into Eq. (2) followed by certainty from a single type of AFM experiment would again be
double integration with respect to x provides the standard forms indeterminate, as with the above case of changing cross section.
for the bent beam profiles as a function of applied load, re- We have used AFM to study polymer nanowires suspended
presentative of AFM force-distance profiling methods: across a ridged substrate [17]. We applied a force at discrete
points down the length of the suspended nanowire sections and
Fb2 x3 Fab2 x2 used Eqs. (5)–(7) to calculate effective elastic modulus. Local
EIz ¼ − ð2a þ L Þ þ ; x V a; ð5Þ
6L3 2L2 indentation was considered to be additive to the bending mode
and was merely subtracted as an offset as a first-order approxi-
Fb2 x3 Fab2 x2 mation; computational work is on-going to determine the ac-
EIz ¼− ð 2a þ L Þ þ curacy of this assumption. The deflection profile generated
6L3 2L2 ð6Þ
F
3 during the measurements seem to validate the assumption of
þ x−aÞ ; xza: fixed ends; that is, the shape of the bending nanowire is better
6
fitted with clamped ends than with simply supported ends. Wu
Relationships of these and similar forms can be used to et al. [16] have extended this method into the study of yield
calculate Young's modulus directly, assuming elastic behavior strength and nanowire plasticity of gold nanowires. Our own
for classic mechanical comparisons. For nanomechanics, values investigations on silver nanowires have shown only elastic
calculated for E are currently interpreted as “apparent” elasticity behavior.
since we have yet to investigate nanoscale bending in full depth. Wong et al. [39] conducted experiments with only one fixed
Boundary conditions are determined by the effect of the support end, while the balance of the wire rested on the substrate, free to
scheme on the shape of the profile constructed from deflection slide in a sweeping lateral motion (Fig. 3). Scanning the wire
data versus location on the nanowire [17]. with AFM in contact mode, they recorded lateral forces at
Several papers [16,34,37] include the following equation,
which is valid at the midpoint for a mid-beam point load.
Letting a = b = x = L/2 yields E = FL3/192zI. Alternately, we can
substitute a known spring constant of a beam, e.g., nanowire,
and calculate its elasticity directly:

kw L3
E¼ : ð7Þ
192I

The slope of an AFM force–distance curve taken against a

surface is the observable spring constant of the mechanical
system, ksys, composed of the cantilever probe being used and
the material under test, which is ideally characterized by the
cantilever and the (nanowire) sample being springs in series:
1 1 1
¼ þ :
ksys kc kw

We obtain kc from a calibration cycle [21,22,43,44], and we Fig. 2. Comparison of deflection profiles for simply supported beam (solid) and
obtain ksys from the slope of the force–distance curve over a beam with clamped or fixed ends (dashed). Units are normalized.
 J.R. Withers, D.E. Aston / Advances in Colloid and Interface Science 120 (2006) 57–67
Fig. 3. Lateral AFM scanning, overhead view of single fixed-end nanowire scan.
various positions along the length of the beam. Their data
indicated that the wire returned to its original position after its
release by the AFM tip, suggesting that the deflection was
elastic and the effects of wire-surface friction were not ob-
served. Lateral forces increased linearly once the tip made
contact with the nanowire, following Eq. (1) to yield a can-
tilever-like spring constant. Further, the slopes of the lateral
force–distance curves, k(x), decreased for scans recorded at
increasing distances, x, from the fixed end. The k(x) versus
x curve qualitatively agreed with the expression k(x) = 3EI/x3
[39]. An effective elastic modulus can be determined from any
force curve, but Wong et al. considered them in toto for a more
general calculation.
3.2. Nanosprings
In 2001, researchers at the University of Idaho synthesized the
first coiled nanosprings [45] with both coil and wire diameters of
the order of tens of nanometers. Currently, AFM mechanical
investigations of silicon oxide nanosprings are underway; while
the measurements have become almost routine, their interpreta-
tion with respect to boundary condition uncertainty remains a
difficult issue. Various other one-dimensional objects similar to
these nanosprings have come under study. Silicon springs of
much larger pitch and coil diameter were reported to exhibit an
electromechanical response to conducting mode AFM [15]; these
long-axis measurements are prone to simultaneous compression
and bending, which can lead to indeterminate conclusions about
material properties. Chen et al. [37] describe a method in which a
nanocoil is clamped between two cantilevered AFM tips–one
with a very compliant cantilever, another of an order of magnitude
higher–by electron beam-induced deposited (EBID) residual
hydrocarbons in a SEM environment. In this case, the force
applied to the spring is determined from the observed deflection
of the more compliant cantilever, and the nanocoil elongation is
determined, both using SEM. A spring constant for the coil is then
found by dividing the applied load by the total elongation, as in
Eq. (1). They assumed that the hydrocarbon “glue” does not
deform, viz., fixed ends.
Bend tests may be conducted for nanosprings, as with the
nanowires, to find Young's modulus. As long as the AFM tip is
not located at either end of the spring, the force applied can be
expressed with simple geometric relationships [46]: F = k
61
(Δ1sinθ1 + Δ2sinθ2), where F is the applied load, k is the
nanospring constant, Δ1 and Δ2 are the elongations of the spring
segments to either side of the applied load beyond their relaxed
states, and θ is the angular displacement of the spring segments,
as shown in Fig. 4.
If we let the tip act at the spring's midpoint, employ appro-
priate trigonometric identities, and assume uniform geometries
and material properties for the length of the spring, the equation
may be written explicitly as a function of vertical displacement
only:
0 1
B L=2 C
F ¼ 2k z@1− qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiA; ð8Þ
2
ðL=2Þ þ z 2
where L is the linear distance separating the spring's two
anchored ends. The bending spring constant, k, can be ex-
pressed in terms of the physical geometry and properties of the
spring material:
Gd 4
k¼ ; ð9Þ
8D3 N
where d is the wire diameter, D is the nanospring coil diameter,
N is the number of involved nanospring coils, and G is the shear
modulus of the material ideally related to elastic modulus [47].
Substituting Eq. (9) into Eq. (8) yields the following result:
0 1
Gd 4 B L=2 C
F¼ @1− qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiAz: ð10Þ
4D3 N 2
ðL=2Þ þ z2
All values may be observed with AFM and/or SEM with the
exception of the shear modulus, which may be estimated from
the ideal relationship E = 2(1 + ν)G, where ν is Poisson's ratio
for the spring material. For transverse loading on the spring, the
relationship between F and z is not expected to be linear. Under
load within the elastic limit, the nanospring wire is twisted rather
than stretched axially in the more usual testing geometry for a
coiled spring. It is not subjected to the same effective loading in
the way its nanowire analog would be in the three-point bend test.
Another complication is the determination of Poisson's ratio.
Values are tabulated for common bulk materials, but may in fact
Fig. 4. Lateral view of nanospring AFM bend test.
62 J.R. Withers, D.E. Aston / Advances in Colloid and Interface Science 120 (2006) 57–67
Fig. 5. Sample nanoindentation force–displacement curve for elastoplastic
loading and unloading.
vary over a sample surface and are completely unknown when
working with new materials, adding another dimension of un-
certainty to the mechanics of nanosprings.
4. Nanoindentation
Nanomaterials find increasing use in modern technologies
such as thermal or protective coatings, dielectrics, nonlinear
optics [48], data storage technologies [49], etc. Mechanical
properties–such as high elasticity–are indicators of successful
long-term material performance, making it important to be able
to determine these characteristics quickly and reliably for many
different length scales. One of the most common and direct ways
of probing local mechanical properties of films or other extended
surfaces is by nanoindentation [48,49], which is most common for
objects that are not suspended. This method employs a sharp, rigid
tip to probe a sample, recording applied force and penetration
depth. AFM can be used as a nanoindenter, but specialized depth-
sensing instruments are also commercially available; some re-
search groups construct their own [49]. At present, the most
common techniques for measuring elasticity with high spatial
resolution are indentation [50–52] and various force modulating
methods with AFM [17,53–56].
A host of nanoindentation methods are available, employing
various tip shapes and relying on different model assumptions
[57–62]. While most, if not all, models were developed in some
fashion from the Hertz contact problem in the late 19th century
[3,8,63], currently applied methods and advanced developments
for indentation modeling are more directly connected to Sneddon's
“major contribution” in this area [63,64]. Hertz analyzed the
problem of the elastic contact between two spherical surfaces with
different radii and elastic moduli. His model assumes elliptical
contact area, frictionless contact, small contact area compared to
the dimensions of each body, that each body can be approximated
as a semi-infinite half-space (i.e., no bulk deflections), and that
deformations occur within the elastic limit. In 1971, Johnson et al.
[65] proposed corrections to this model to account for adhesive
forces between the bodies in what is now called the JKR theory.
Still, most nanoindentation methods require corrections to these
idealized models. Further corrections on the initial contact regime
are also available, having more pertinance in adhesion studies
rather than indentation [20,66,67]. The Oliver and Pharr technique
is commonly used after initial indenter contact [57,58,68–71],
extending from prior research conducted by Sneddon [64] and
others [72–74].
Nanoindentation collects data from a complete loading and
unloading cycle to generate a particular force–distance curve
(Fig. 5), very similar to an AFM force profile. Force versus
penetration depth (or displacement) is desired instead of the force
versus separation of typical AFM force spectroscopy or nanowire
bend tests. In the case of a purely elastic deformation, there will
be no hysteresis between the loading and unloading curves. Con-
sideration of plasticity or viscoelasticity is a much more complex
problem; much of our understanding in this area is the result of
experimentation and finite element simulation [63].
Oliver and Pharr consider the cantilever-surface system as a
two-spring system, as above. The total measured displacement
is a combination of the load frame (AFM cantilever) dis-
placement, hl, and that of the sample, hs: htot = hl + hs. We con-
vert this expression to compliances by dividing the force, F,
giving effectively the same relationship as that for the spring
constants in series described above for bending: Ctot = Cl + Cs,
where compliance is simply the inverse of the stiffness, S (C = 1/S).
This is analogous to bending but characteristically different in the
actual process of indenting. Cs is given by
pffiffiffi
p 1
Cs ¼ pffiffiffi : ð11Þ
2Er A
A is the contact area, and Er is the effective, or reduced, Young's
modulus of the system given by
1 1−m2s 1−m2i
¼ þ : ð12Þ
Er Es Ei
Eq. (11) holds for any indenter that can be described as a
body of revolution of a smooth function, and it is not limited to a
specific geometry. Oliver and Pharr suggest an iterative scheme
to determine Cl and A, where an initial estimate for a perfect
Fig. 6. Nanoindentation schematic of indenter tip and surface.
 J.R. Withers, D.E. Aston / Advances in Colloid and Interface Science 120 (2006) 57–67
Berkovich tip is A ≅ 24.5hc2: hc is the contact depth (Fig. 6),
given by hc = hmax − hs; hmax is measured at the peak load, and hs
can be determined from the force–penetration data, hs = εFmax/S;
ε varies with indenter geometry, as in Table 1. The factor of 24.5
is not accurate for other geometries, and especially for AFM
indentation, the individual tip in use must be independently
characterized with electron micrographs or one of the standard tip
imaging deconvolution techniques [19,75–80]. Commercial
AFM probes now come with a variety of comparably sharper
tips with high aspect ratios such that the contact mechanics may
be substantially different from classic indentation experiments. In
any event, using higher-order fits for the area–indentation rela-
tionship (A ∼ hc2) is theoretically dubious and yet may provide
some practical improvement in data correlation.
Initial estimates of Cl and Er are obtained by plotting Ctot
versus A1/2 for several indentations made in a standard reference
sample, such as aluminum. Revised calculations of A will alter
estimates of Cl and Er, so the process is repeated until con-
vergence. An alternate method of finding A is to image the
plastic impression with AFM [70]. For a highly plastic reference
material, this should give about the same projected area as the
contact area at maximum load. Again, plotting Ctot against A1/2
yields Cl and Er from the slope and y-intercept of a linear fit.
Once this quasi-calibration method is complete, we observe
the force-penetration curves for our samples of interest. The
unloading portion of the curves is fitted to a power law ex-
pression: F = α(h − hf)m, where the parameters α, hf, and m are
all determined by a least squares fitting procedure of the curve;
hf is the final unloading depth; m is related to the geometry of
the indenter; α accounts for geometric constants, the sample
elastic modulus, the sample's Poisson ratio, the indenter elastic
modulus and the indenter's Poisson ratio [57,60]. The stiffness
is found by analytically differentiating this expression and
evaluating the derivative at the peak load and displacement,
Fmax and hmax:
S¼
dF
dh
jhmax ;Fmax
¼ amðhmax −hf Þm−1 :
Doerner and Nix [74] took their initial unloading slope to be
linear, assuming the contact area remained constant. Oliver and
Pharr [63] demonstrated that the unloading slope is never linear
in practice; the contact area changes immediately and contin-
uously during unloading. The Oliver and Pharr method predicts
actual behavior reasonably well, within 4% of values reported in
the literature for materials with isotropic elastic properties [70].
There are some difficult points to consider in the use of this
model. Error is more pronounced in materials that show a large
elastic recovery [58]. To compute the modulus from the mea-
Table 1
Typical values for ε and m
Indenter geometry ε m
Flat-end cylinder 1 1
Parabaloid 0.75
Cone 0.73
63
sured values of Er requires that Poisson's ratio be known. As we
will discuss later, when the ratios are not known, we can map
only relative material property differences for the sample. Also,
the validity of this approach depends on there being an ideal
indenter geometry or, in the worse case, precise modeling of the
real tip shape, which is computationally intensive.
Indentation testing is very common in the determination of
elastic modulus and hardness of supported thin films
[49,51,68,71,81,82], which introduces the added complication
of substrate influences [81] due to finite sample thickness
[54,56] and/or the presence of multiple layers [83].
4.1. Living cells
Extensive progress has been made over the years in under-
standing the surface properties of microorganisms using a variety
of methods [84], such as micropipette aspiration, dynamic
reflection interference contrast spectroscopy, scanning acoustic
microscopy, infrared laser traps, and various magnetometric anal-
yses. Most of these methods average properties over compara-
tively large areas with only modest spatial resolution.
It is relatively straightforward to create topographic images of
a cell sample using AFM [85,86]. Determination of its mechanical
properties can be accomplished using a nanoindentation process
like that discussed above, even under actual physiological con-
ditions [85,87,88]. Force curves are recorded and converted into
force-indentation plots using appropriate models. These profiles
can then be analyzed to give quantitative information about the
surface, such as E. Nanoindentation has been accomplished on
animal cells including glial cells, platelets, cardiomyocytes, ma-
crophages, endothelial cells, epithelial cells, fibroblasts, osteo-
blasts, and more [33,87,89–95]. AFM force measurements have
also been applied to microbial specimens [96], resulting in
spatially well-resolved maps of mechanical properties [33,97].
Understanding biomechanical mechanisms may one day be key in
strengthening bone, growing cartilage, improving cardiac con-
tractility, and constructing tissues for artificial organs [98].
Hertzian-based models are still commonly used in the
evaluation of elastic modulus [84,88,94,97,99]. Several rela-
tionships have been published for applied force vs. tip
penetration depth, varying again with indenter shapes. For ex-
ample [94,97], the conical and paraboloidal tips would be of the
following forms:
2
Fcone ¼ tanaEr d2 ;
p
4
Fparaboloid ¼ Er R1=2 d3=2 :
3
In the case of soft samples and sufficiently rigid probes, Eq.
(12) reduces to
Es
Er ¼ :
1−ʋ2s
1.5 These models have been demonstrated to be adequate in
2
many applications even though cells do not meet the model
64 J.R. Withers, D.E. Aston / Advances in Colloid and Interface Science 120 (2006) 57–67
assumptions. In particular, living cells are anisotropic and will
not necessarily behave elastically without a viscous component,
or loss modulus [95]. Hertzian models are not appropriate where
adhesive forces are present. Another difficulty is that Poisson's
ratio is not measured in the indentation experiment. In practice,
it is often arbitrarily assigned the constant value of 0.5 (the case
for incompressible bodies), and we would expect sensitive
stiffness measurements across the surface of a cell to resolve
real property variations in local deformation because of inter-
cellular heterogeneities.
Other sources of error arise from characteristics of the AFM,
which are issues in any quantitative experiment. Choice of can-
tilever spring constant and accurate calibration thereof (see
Section 2) are primary examples. If a sample is soft relative to
the sensitivity of the AFM, the sample will deform in response to
the tip before the cantilever deflects measurably and the true
contact point will not be detected. Large uncertainty in the contact
point leads to significant errors in the estimation of the indentation
depth, elastic moduli, or any other parameter defining the contact
behavior [33,84,93]. The soft cellular matter requires an equally
soft cantilever. The wide range of AFM tip shapes and the small tip-
sample contact area make the contact geometry difficult to define.
Dimitriadis et al. [99] write that the error introduced into the
calculated elastic modulus by the aggregate of tenuous assump-
tions can be an order of magnitude. They derive corrections to the
Hertz model that cover the full range of contact force, tip radius,
and sample thickness. In many instances–even in materials other
than cells–variations of stiffness are more interesting than quan-
tifying elasticity, as indicated by Heinz and Hoh [33,87]. Studies
of spatial and temporal changes in mechanical properties on a
relative scale can lead to many insights without a calibrated
standard for Young's modulus. In that case, these significant
errors in absolute property measurements are no longer important.
4.2. Dendrimers
The first hyperbranched molecule was patented in 1987
[100]. Its architecture immediately interested synthetic che-
mists, but it took some time to attract the mainstream, possibly
because of difficulty in characterization due to irrational solu-
bility and irreproducible molecular weight. Most of the research
effort has come from industry.
Hyperbranched molecules manifest unique properties as a
result of their intrinsic globular structure and large number of
terminal functional groups. Unlike dendrimers, which are “per-
fectly” branched, hyperbranched polymers have elements of
conventional polymers, namely molecular weight polydispersity,
isomerism, and geometrical shapes. A normalized branching
factor was introduced to describe the degree of perfection of the
branching [100,101]: fbr = (T + B)/No, where T is the mole fraction
of monomers at the terminal position, L is the number of un-
branched monomer units, B is the number of fully branched
monomer units, and No = T + B + L. Thus, the branching factor for
a fully branched polymer is one, such as a dendrimer, and fbr of a
linear polymer is zero.
In practice, dendrimers and hyperbranched polymers are
usually discussed together using the term dendritic polymers.
Hyperbranched polymers are often more rapidly prepared and
more economical than ideal dendrimers for certain applications.
The high symmetry of perfectly branched dendrimers, their
cascade synthesis method, and the lack of a molar mass dis-
persity (assuming ideal growth) show a closer similarity to
biomolecules like DNA and polypeptides rather than classical
polymers [101].
Study of hyperbranched molecules has increased in recent
years, with about 2000 publications in 2004 [102]. The field is
too new to draw conclusions about the ultimate usefulness of
dendritic polymers and far too broad to review current efforts
completely here. It is widely believed that they offer the chance
for the development of new products, but their complexity and
broad variety of structures present a monumental challenge.
It is thought that certain functional groups can be placed at
the sterically congested core of dendritic polymers, giving them
highly selective reactivity [103,104], although lately, the inter-
est seems to have turned more to studies of surface functional
group behaviors [105–108]. Dendritic polymers may also find
application in medicine, catalysis [104], or nanoelectromecha-
nical systems (NEMS) [109]. AFM has been used to create
topographical images of dendritic polymers [110], and AFM
chemical force modes have allowed the study of reactivity at the
peripheral functional groups including branch termini [24,111].
Researchers at Iowa State University [112] pursued mechan-
ical studies of dendrimers similar to those used in the study of
living cells, using Hertzian contact models. Force–distance data
showed that compression behavior varied under low and high
normal loads with large initial deformation followed by a stiffer
response at higher applied forces. This is consistent with
behavior of a compliant sample on a rigid substrate. It appeared
that higher-generation dendritic molecules possessed a much
higher elastic modulus, due to higher internal molecular stiff-
ness and a crowded periphery. As for cells, indentation studies
of dendrimers and the like are more complicated than nano-
indentation on flat films and other extended surfaces.
5. Liquid interfaces
Liquid interfaces of gas bubbles and droplets demonstrate
some resistance to deformation under an applied load, exper-
imentally similar to elasticity measurements. As such, we
observe effective spring behavior in the fluid interfaces, which
may be deconvoluted to quantify various interfacial phenomena
such as colloidal interactions or local surface tension. Much
effort has been expended in the attempt to characterize this
behavior, with some discussion as to the suitability of Hookean
spring models. Studies have examined the interaction of
bubbles/droplets with colloid particles [29,112–119] as well
as the interaction between two liquid particles [120]. Unlike
solid surfaces, interaction models for fluid interfaces do not
usually require a Born repulsion term in the net interparticle
force relationship since physical contact with a fluid is a wetting
condition [20,114].
To a greater degree even than in studies of cells or polymers,
a significant stumbling block to force measurements on highly
deformable systems is that there is no clear transition from
 J.R. Withers, D.E. Aston / Advances in Colloid and Interface Science 120 (2006) 57–67
noncontact to contact [33,119]. Surface deformation occurs
prior to contact due to the extended range of surface forces and
fluid dynamics. Thus, the zero of separation cannot be deter-
mined with AFM in the same way as for rigid bodies.
Gillies et al. [119] summarize a variety of ways in which
researchers have defined zero separation. In some cases, the
scanner position is recorded, and all separations are considered
relative to an arbitrary zero [121,122]. In others, deformation is
modeled as a Hookean spring and contact is considered to be the
point at which the linear compliance curve reaches zero force,
either upon approach or during the retracting of the probe,
which is only valid if the surfaces come into intimate contact
[123–125]. Others take the zero to be where deformed surfaces
in contact exhibit zero force. This occurs due to a balance of van
der Waals attraction and steric (or other) repulsions, and the
amount of deformation can be substantial [126–128]. The zero
of separation is sometimes taken to be that corresponding to the
maximum force. This approach also ignores the flattening of the
deformable bodies and can be in serious error [29,114,129,130].
Contact is sometimes taken to occur at the point of the first
measurable force. This is appropriate for contact forces of
essentially “zero range” but not for long-range forces such as
those of the electrostatic double layer. Burnham et al. [131]
defined contact as the position where repulsion is first detect-
able, indicated by a change in curvature of the force-distance
profile, i.e., a break point. A related method fixes the zero of
relative separation at an arbitrarily small force [132], which can
serve to eliminate the uncertainty of defining the onset of
deviation from the baseline of no interaction, defined at large
probe-sample separations. Mulvaney et al. [115] fitted the
nonlinear Poisson-Boltzmann equation for rigid bodies by
adjusting both the zero of the experimental separation and the
surface potential. Each of these approaches has its limitations; in
order to have meaning, zero separation or contact must be
interpolated from experimental data in some self-consistent
manner. In all events, the definitions of the distance or sepa-
ration origin are made for experimental and/or modeling con-
venience and are of limited theoretical use. There is yet no way
to control a fluid–fluid interface directly for all separations of
interest.
Colloid probe force measurements have demonstrated
Hookean spring behavior in air bubbles and oil droplets in the
constant compliance region. Theoretical work [113] has con-
firmed this experimental finding, showing that the experimen-
tally applied loads are well within the linear region, and that the
effective spring constant of a fluid interface, keff , is practically
equal to the interfacial tension [132]. However, this is highly
dependent on geometry and scaling. It is suspected that this
would only be the case within some limiting regime, like that of
small interfacial deflections.
If the interface were to behave as a Hookean spring, then it
should be possible to convert the force versus total distance
displaced into the true force-separation profile simply by adding
the quantity of force divided by keff: separation = distance + F/keff.
The quantity F/keff is the interfacial displacement normally
inferred from AFM experiments on air bubbles or oil droplets.
The value of keff is the slope of F(z) at high load, considered to be
65
similar to hard wall contact with a solid surface. But keff is
different depending on where it is measured; greater deformation
yields a larger linear spring constant. Both experiment and theory
point to a nonlinearly deflecting fluid–fluid interface, though
there are certain experimental circumstances under which a linear
approximation is very accurate [118].
Furthermore, this provides a possible explanation for the
apparent transition observed by Hartley et al. from the expo-
nential force behavior of overlapping double layers, when the
drop acts like an ideal spring switching to the “simple linear
relationship” of force with apparent separation. This is not the
same as linearity of the interfacial deformation with load. To
maintain the analogy, if the oil–water interface is like a spring
but with an increasing stiffness, then keff may be expressed as a
weak function of drop deflection. It might be useful to fit
experimental data with a power function, as is common for
indentation, to convert F(z) curves into F/R vs. separation: R is
the system radius of curvature, used to normalize data [20];
F = kdn, where d is the drop deflection, k is constant, and n is
slightly increasing as d increases (values of n taken from fitting
the results). If the stiffness is indeed equivalent to the interfacial
tension, then the dn term is merely the z-component of a surface
integral of the interfacial curvature, which has no analytical
solution. It remains to be shown that physical conditions, if any,
would lead to this behavior. Empirically, there seems to be a
connection between the onset of the drop dimpling inward and
the deviation from linear interfacial deflection, that is, the drop
apex curvature inversion, from convex to concave. It is likely a
geometric consequence of measuring only the normal force
component of a curved film with increasing interfacial area
taking part in the force measurement [29,114,133].
6. Concluding remarks
The advent of AFM was of great import to nanomechanics.
Phase contrast, pulsed-force and other intermittent contact modes
provide high spatial resolution of surfaces, highlighting inhomo-
geneities and relative surface property differences. Instrumenta-
tion and modeling have matured–and are constantly evolving–to
where they offer a useful look into nanoscale mechanical per-
formance. Now, nanomechanical investigations of molecular
changes that precede a material yielding, creeping, crazing,
cracking, or otherwise failing at a macroscopic level are feasible.
One continuing problem with the determination of absolute
force data lies in knowing Poisson's ratio. In general, this is
obtained from tabulated data for known, bulk materials, or it is
given an assumed value, as in the study of cells. Another problem
is that the tip-sample contact area is difficult to quantify, which
can lead to large errors. Still, relative, local surface properties are
easily mapped, and absolute, quantitative data are unnecessary
for many experiments and applications in any case.
The true challenge will be to determine the local effects of
making nanomechanical measurements. The authors are cur-
rently investigating energy loss in polymer nanowires with
bending and nanoindentation experiments to determine how
AFM alters molecular conformations. Preliminary studies show
accelerated aging across repeated deformation experiments,
66 J.R. Withers, D.E. Aston / Advances in Colloid and Interface Science 120 (2006) 57–67
suggesting that a kind of strain hardening or crystallization is
taking place. The fundamental reasons for increased apparent
elastic modulus in nanostructures remain unproved. Even
though significant obstacles remain, the collection of recent
advancements in nanomechanical measurements of material
strength are laying a strong foundation to improve our under-
standing of basic material behavior such as beam bending and
plastic deformation.
Acknowledgements
The authors gratefully acknowledge partial support for this
work from the National Science Foundation (EPS0132626,
EPS0447689) and the W.M. Keck Foundation.
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