The new future of scanning probe microscopy: Combining atomic force microscopy with other surface-sensitive techniques, optical microscopy and fluorescence techniques

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The new future of scanning probe microscopy: Combining atomic forcemicroscopy with other surface-sensitive techniques, optical microscopyand fluorescence techniques

Susana Moreno Flores* and Jos�e L. Toca-Herrera*

Received 24th June 2009, Accepted 10th August 2009

First published as an Advance Article on the web 3rd September 2009

DOI: 10.1039/b9nr00156e

Atomic force microscopy (AFM) is in its thirties and has become an invaluable tool for

studying the micro- and nanoworlds. As a stand-alone, high-resolution imaging technique and force

transducer, it defies most other surface instrumentation in ease of use, sensitivity and versatility.

Still, the technique has limitations to overcome. A promising way is to integrate the atomic force

microscope into hybrid devices, a combination of two or three complementary techniques in one

instrument. In this way, a comprehensive description of molecular processes is at hand; morphological,

(electro)chemical, mechanical and kinetic information are simultaneously obtained in one experiment.

Hereby we review the recent efforts towards such development, describing the aim and the applications

resulting from the combination of AFM with spectroscopic, optical, mechanical or electrochemical

techniques. Interesting possibilities include using AFM to bring optical microscopies beyond the

diffraction limit and also bestowing spectroscopic capabilities on the atomic force microscope.

AFM: blindly probing surfaces may not be enough

Deprived of our eyesight, we humans sense the objects around us

with the aid of our hands or sticks. In this way we get an idea

about objects’ size, their shape, their texture and their hardness.

As we step forward, we intuitively probe our immediate envi-

ronment by systematically moving our hands or swinging a stick

to the left and right, and up and down. We register the position of

Biosurfaces Unit, CIC biomaGUNE, Paseo Miramon 182, San Sebastian,Spain. E-mail: [email protected]; [email protected]

Susana Moreno Flores

Susana Moreno Flores gradu-

ated in Chemistry from the

Complutense University,

Madrid. This was followed by

her doctorate on polymer

dynamics using dielectric relax-

ation spectroscopy at the same

university, which she finished in

2001. Between 2001 and 2003

she did postdoctoral research on

the surface properties of poly-

electrolyte monolayers at the

Ecole Normale Sup�erieure,

Paris. Between 2003 and 2006

she was a postdoctoral fellow at

the Max-Planck Institute of Polymer Research, where she gained

experienced with scanning probe microscopies in the group of Prof.

H.-J. Butt. In 2007 she joined the group of Jos�e Luis Toca-Herrera

at CIC biomaGUNE.

40 | Nanoscale, 2009, 1, 40–49

an obstacle and we follow its contours by just sliding or tapping

our fingertips over its surface.

The atomic force microscope certainly resembles the human

analog as a blind microscope that can sense micro-and nano-

objects (Fig. 1). Indeed, the instrument provides the probing

stick, a microsized cantilever with a tip at its free end; a piezo-

driven device to move the probe over the sample (or vice versa) in

three dimensions with nanometre precision; a means to get the tip

position during its movement and a feedback mechanism to

control how strongly the tip slides or taps over the sample

surface. In this way, atomic force microscopy (AFM) has become

an invaluable technique to explore the morphology of the

Jos�e L: Toca-Herrera

Jos�e Luis Toca-Herrera gradu-

ated in Physics from the

University of Valencia, in 1993.

In 1994 he did one-year research

at Max-Planck Institute of

Polymer Research. From 1996

to 1999 he completed his PhD at

the Max-Planck Institute of

Colloids and Interfaces under

the supervision of Helmuth

M€ohwald. He has held Post-

doctoral positions at the Tech-

nical University-Berlin, the

University of Cambridge, and at

the Center for Nano-

biotechnology (BOKU-Vienna). In June 2004, he joined the

Rovira i Virgili University as Ram�on y Cajal Researcher. In 2007

he moved to CIC biomaGUNE as group leader. Since June 2008 he

is I3 research professor.

This journal is ª The Royal Society of Chemistry 2009

Fig. 1 AFM imaging basics as a blind microscope analog. By blindly inspecting an object just underneath we systematically move our body and stick

right and left, and up and down. We can either slide or tap our stick over the surface. Our brain controls the movement and the force we exert on the

surface through our hand and stick. AFM resembles the analog with a cantilever as an elastic stick, a piezoscanner that moves it along three dimensions,

a laser, and a position detector that registers its position. The ways the tip can scan the surface are more numerous than we humans have, however, on

most occasions the tip is either made to slide over the surface, impinging a defined force (contact mode imaging) or to tap the sample more or less gently

(intermittent contact imaging). The operation is electronically controlled to ensure the scanning is being executed under the desired scan conditions.

Finally, a computer processor presents the result as an image. (From ref. 31).

nanoworld. Moreover, since the cantilever is a force transducer,

it has been extensively used to study surface and molecular

interactions.

In spite of its many advantages, the atomic force microscope

as a stand-alone tool retains some limitations. Spectroscopic

identification is beyond its reach, as are determination of contact

areas or absolute distances between probe and sample.

Combining the atomic force microscope with complementary

techniques to build hybrid instrumentation promises more than

one way to overcome these limitations. Synergy may result from

such combinations. Optical microscopy bestows on AFM the

eyes for precise tip positioning in microstructured materials or

cells; interferometry confers the capability of measuring absolute

distances, and Raman spectroscopy, the label-free chemical

identification of species. Reciprocally, AFM coupled with

model-based techniques such as ellipsometry, surface plasmon

resonance or even quartz crystal microgravimetry is of invaluable

assistance in model validation, the calculation of adsorbed mass,

the estimation of trapped solvent or assessing the effect of sample

heterogeneity. We will briefly summarize the state-of-the-art

developments of such combined techniques.

AFM + optical microscopy

Applying light microscopy to the blind atomic force microscope

has turned out to be a great breakthrough in the applicability of

the latter, especially in the field of biosciences. In particular, the

combination of the two techniques has made it possible to

investigate cellular processes in an unprecedented way, defying

conventional biological practice.27 With the aid of light micros-

copy, precise positioning of the AFM tip into the region of

interest of microsized objects such as particles or cells is possible,

especially after the introduction of technologies that allow the

accurate overlay of optical images and tip lateral displacements.

Using transparent substrates, AFM imaging has been combined

with the family of transmitted light optical microscopies such as

phase and differential interference contrast (DIC), fluorescence,

confocal laser scanning microscopy (CLSM), total internal


reflection fluorescence (TIRF) and fluorescence lifetime imaging

microscopy (FLIM). Recent implementation of AFM with

upright microscopes has enabled the extension of the application

of the combined techniques and epifluorescence to non-trans-

parent substrates and hence to a wider range of materials.28 Last

but not least, micro- and nanointerferometric techniques based

on transmitted light microscopy such as reflection interference

contrast microscopy (RICM), are particularly useful when

combined with AFM. Indeed, RICM provides complementary

information such as absolute tip–substrate distances and contact

areas, which the AFM as a stand-alone device cannot produce.

Modification of the AFM setup to be coupled to an inverted or

an upright microscope requires the creation of a obstacle-free

optical path to the AFM cantilever and the substrate to be

analyzed (Fig. 2). Thus, the configuration of cylindrical piezo-

scanners mounted upright on tips or on samples is avoided. In

particular, tip-scannable atomic force microscopes are preferable

when simultaneous optical and AFM imaging is required. In the

case of inverted microscopes, a considerable effort in AFM

design is needed so as to accommodate optical condensers of high

numerical aperture (0.55) without compromising the quality of

optical imaging or instrument stability.

AFM-CLSM

Combining confocal and atomic force microscopy on fluo-

rescently labelled samples has become a unique tool in imaging

molecular complexes on cellular surfaces at high resolution.27

The confocal microscope makes use of coherent laser illumina-

tion and an optical pinhole system to extract fluorescence images

from different focal planes. As a result, the sensitivity and image

quality is very much improved with respect to conventional

fluorescence microscopy. Cell organelles can be selectively

labelled using standard immunolabelling, and colocalized with

the 3D topological images provided by the atomic force micro-

scope (Fig. 3, left). Morphological studies on focal adhesion

structures are an example of a successful combination of high-

resolution imaging with fluorescence mutilabelling to ascertain

Nanoscale, 2009, 1, 40–49 | 41

Fig. 2 Left: Scheme of the combination of a transmitted light inverted optical microscope with the AFM. To ensure simultaneous optical and AFM

imaging, the AFM tip is allowed to move in three dimensions on a static stage. Right: Optical and AFM image overlay of a breast cancer cell cluster

(MCF7). Bar ¼ 25 mm. Vertical scale of the AFM image from grey to white: 10 mm (author’s contribution).

the localization of interacting molecules within protein

complexes12 (Fig. 3, right).

AFM-TIRF

TIRF profits from the evanescent wave generated at a surface by

a totally reflected light source. This evanescent wave, which is

physically identical to that which excites plasmons on metallic

surfaces, extends a few tens of nanometres along the surface

normal. The wave is capable of exciting fluorophores, which is of

particular advantage, since only those in close proximity to the

substrate can be excited. As a consequence, a fluorescence image

of the immediate vicinity of the substrate can be obtained.

An interesting application of the combined AFM-TIRF

technique has been applied to live cells. Cells adhere to affinity

substrates by developing so-called focal adhesion points,

arrangements of proteins that act as anchors. TIRF microscopy

(TIRFM) can map the location of these adhesion points while

the AFM cantilever exerts forces on certain cell positions. In this

Fig. 3 Left: Combined AFM and confocal imaging of mouse embryonic fibro

imaging of cells exhibiting fluorescently labelled organelles (blue, nuclei; green

(reproduced from ref. 27 with kind permission from Springer Science + Busine

roofed cells, with actin labelled in red (A) and YFP-paxillin (B, green). The pa

labelled confocal image, but not in the paxillin image, indicating that paxillin lo

beyond in the membrane-distal half. (From ref. 12, reproduced with permiss

42 | Nanoscale, 2009, 1, 40–49

way, force transmission from the apical membrane to the basal

membrane of cells has been detected from variations in the

number and arrangement of focal adhesion points in TIRFM

images.29 At the molecular level, TIRFM and AFM have been

successfully correlated to ascertain the morphology of myosin

self-assembled filaments in a recent study3 (Fig. 4).

AFM-FLIM

This promising combination of techniques has been rather

poorly exploited, although it was first reported and applied

in 2002.17,22 The setup shares common features with that of

AFM-CLSM,17 and it has mainly been applied to fluorescent

nanospheres, labelled DNA17 and live bacteria.30 Lifetimes of

fluorophores attached to molecules or spheres can, however,

be altered, which makes the interpretation of data

particularly difficult. In particular, the presence of the AFM

tip—typically Ag or Au metal-coated—can either induce fluo-

rescence quenching or fluorescence enhancement, which may

blast. (a) and (b) are AFM topography and deflection images; (c) confocal

: actin filaments; red: clathrin); (d) overlay of confocal and AFM imaging

ss Media). Right: High-resolution imaging of focal adhesion points of de-

rtial disruption of the architecture is shown in the AFM (D) and f-actin-

calization is more proximal to the focal adhesion while f-actin is localized

ion of the Journal of Cell Science).


Fig. 4 Left: TIRFM (a) and AFM (b) images of three miosin filaments.

(AFM image obtained in intermittent contact mode). The inset of (a)

shows the optical microscope point spread function (Gaussian fitting,

standard deviation¼ 260 nm). Right: TIRF intensity as a function of the

height measured by AFM. The intensity is a linear function of the height,

which indicates that the fluorophores, situated at the heads of the myosin

molecules are arranged around the filament, instead of being embedded.

(Reproduced from ref. 3 with permission from Elsevier).

change lifetimes as well as fluorescence intensity. The net effect of

these two opposing factors is not possible to predict to date.

AFM-RICM

Combining these two techniques is especially advantageous when

studying surface interactions and mechanics.15 AFM as a dyna-

mometer detects forces exerted between two interacting surfaces

as they are being approached or separated; the force is quantified

through the cantilever deflection, which behaves as a linear

spring; RICM as an interferometer is capable of detecting the

distance between these two surfaces with a precision on the order

of 1–3 nm. To do that, light is shone on the region between the

AFM probe, usually a glass bead, and the substrate. The light is

reflected at both interfaces, i.e. that between the substrate and the

surrounding medium and that between the medium and the

probe (Fig. 5a, left). Constructive and destructive interference

between the two reflected rays occurs as the tip–substrate sepa-

ration varies. The interference pattern is thus a function of

the tip–substrate distance, and also of the bead contour, the

wavelength of the incident light and the optical properties of the

medium (Fig. 5b, left). Using this combined approach, protein–

ligand interactions,16,39 and microcapsule and droplet deform-

ability9,11 have been verified (Fig. 5, right).

AFM + Raman spectroscopy: tip-enhanced Ramanspectroscopy (TERS)

Introduced at the turn of the 21st century1,38 and in constant

development ever since, TERS reveals itself as a powerful tech-

nique; it combines the high spatial resolution of the scanning

probe microscopies with surface-enhanced Raman spectroscopy

to obtain morphological and chemical information simulta-

neously for surface components at the nanoscale.38

TERS is a near-field microscopy that makes use of an aper-

tureless probe to enhance the Raman signal exerted by a sample.

The probe is a sharp metallic (e.g., Ag or Au) tip, typically


a metal-coated AFM tip or an electrochemically etched metal

wire, that is irradiated along its apical axis by a low-power laser

at a visible wavelength (500–650 nm). When this tip is placed

sufficiently close (approx. 1 nm) to the sample, field-enhance-

ment occurs leading to molecular excitation of the nearby sample

area and thus local Raman spectra can be obtained. The mech-

anism of this enhancement is still an issue under discussion,

however it is agreed to be intimately connected to the tip material

and tip geometry and it is generally believed to have a double-

nature: electromagnetic and chemical.36,38 The electromagnetic

enhancement has to do with the excitation of metal plasmons at

the tip apex by the laser, with the tip acting as an antenna. The

generated field has a reduced spatial range and can only excite

molecules that are 1–20 nm apart in the vertical direction and

20–50 nm apart in the lateral direction. The chemical enhance-

ment (also called charge transfer) is based on molecular inter-

actions between the metal tip and the sample surface that alters

the spectroscopic properties of the latter. In this regard, TERS

presents an additional advantage with respect to the classical

bulk or surface-enhanced Raman spectroscopy (SERS) since

Raman spectra can even be obtained from poor Raman scat-

terers, chemical specimens that otherwise exhibit very low or

undetectable Raman signals.

The most common TERS setups are shown in Fig. 6. TERS

can be operated either in reflection (especially convenient for

non-transparent samples, Fig. 6a)33,34 or in confocal mode

(Fig. 6b),36,41 which refers to its combination with optical

reflection and confocal light scanning microscopes, respectively.

In both cases, a p-polarized laser is focussed on the tip so that the

polarization plane is kept parallel to the tip axis. Laser focussing

is done by means of an optical objective (usually 50� or 60�)

which is also used to collect the Raman radiation from the

sample. In the reflection version a notch or edge-filter (NF or EF)

is usually placed before the spectrometer to collect only the

backscattered radiation (Fig. 6a). In the confocal version, an

additional beam splitter (BS) and a flipping or folding mirror

(FM) are placed before the spectrometer, which redirect both the

laser source and the sample emission to a photomultiplier. In this

way it is possible to switch rapidly between confocal imaging and

Raman spectroscopy.

TERS has been validated through its application to model

systems such as dye molecules and carbon nanotubes, which both

exhibit characteristic and well-known Raman signals.33,38 As far

as biomaterials are concerned, studies have been accomplished

on nucleic acids and biofilms such as alginates or bacterial

surfaces.36 Recently TERS has been said to be the only technique

suitable for the study at the nanoscale of subsurface features of

thin polymer films41 (Fig. 7, right). An important and common

issue in all these studies is the achievement of a high tip-

enhancement contrast, defined as the ratio of the Raman signal

intensity when the tip is approached, to that when the tip is

withdrawn from the sample33 (Fig. 7, left).

A still pending issue of TERS is finding the optimal tip prep-

aration technique that ensures good reproducibility and high

enhancement.33,41 On the other hand, the application of TERS to

complex biological samples such as biofilms is still cumbersome

work that involves proper spectral interpretation and makes it

difficult to locate characteristic bands that are sample-specific.36

Tip-induced interference, such as carbon contaminations or

Nanoscale, 2009, 1, 40–49 | 43

Fig. 5 (a) AFM-RICM setup. The tip is typically a microsized (10–20 mm) glass bead attached to the cantilever. The incoming light I0 is reflected at the

substrate–medium interface (I12) and at the bead–medium interface (I23). (b) The resulting interference pattern of the glass bead. From the distance and

the intensity distribution of this image is it possible to calculate the glass bead profile and the bead–substrate distance (reproduced from ref. 39 with

permission from Elsevier). (c) Evolution of the interference pattern while an AFM cantilever is approaching, in contact with, and withdrawing from

a substrate (reproduced from ref. 16 with permission from Elsevier). The jumps and discontinuities of the AFM cantilever are visualized in RICM as

fringe displacements. (d) Deformation of a polyelectrolyte multilayer capsule. The interference pattern shows the extent of deformation while the AFM

exerts an ever-increasing force on the capsule (reproduced from ref. 9 with kind permission of the European Physical Journal).

far-field effects usually appear as large background signals and

they should also be properly addressed.

AFM + surface plasmon resonance

Both techniques were reported to be successfully combined in

the middle of the 1990s.5,37 SPR is a technique that can

monitor changes in the optical density of thin films on metal-

coated surfaces through the excitation of surface plasmons

with an incident light source. The light source typically

Fig. 6 Tip-enhanced Raman spectroscopy (TERS

44 | Nanoscale, 2009, 1, 40–49

consists of a laser of 600–700 nm wavelength that, in the most

common configuration, is incident on the noble metal layer

through a prism, at a range of angles that are above the

critical angle where total internal reflection occurs. At

a certain angle, the so-called resonance angle, excitation of the

surface plasmons occurs, leading to a minimum in the

reflected light. The resonance angle shifts whenever the optical

density of the layer in contact with the noble metal undergoes

a change, and thus it allows detection of the adsorption or

desorption of molecules.

) in reflection mode (a) and confocal mode (b).


Fig. 7 Left: Effect of tip-enhancement on the Raman spectrum of Brilliant Cresyl Blue, using (a) a gold-coated AFM tip or (b) an etched bold wire

(reproduced from ref. 33 with kind permission of the European Physical Journal). Blue traces correspond to the withdrawn probe, while red traces to the

engaged probe. Right: Chemical identification of film constituents. (c) AFM topography image of a blend copolymer film composed of polystyrene (PS)

and polyisoprene (PI) showing circular protrusions. (d) Sequence of Raman spectra collected at the positions depicted in (c). (e) Raman spectra of pure

PS and PI. (f) Magnified view of spectrum collected in background positions. Protrusions are shown to be PS-enriched, on a PI-enriched background

(reproduced from ref. 41 with kind permission of the European Physical Journal).

The geometric complementarity of both AFM and SPR

methods greatly eased the issue: the AFM needs to be mounted

on top of the sample surface through a tip 3D-moving config-

uration, while the Kretschmann configuration of the SPR setup

reaches the sample from underneath (Fig. 8, left). Coupling

Fig. 8 Left: Instrumental configuration for AFM + SPR. AFM and the o

requires a Kretschmann configuration. Right: Simultaneous AFM, cyclic vol

a potential cycle from 0 to 1.2 V (reprinted with permission from ref. 2. Cop


both techniques, it was possible to monitor in situ the kinetics of

the hydrolysis of biodegradable polymer films5,37 and more

recently the electropolymerization and deposition of the

conductive poly(3,4-ethylenedioxythiophene) (PEDOT) on

gold electrodes.2 In this work, cyclic voltammetry and

ptical setup reach the sample from above and below, respectively. SPR

tammetry and SPR data during the electropolymerization of PEDOT by

yright 2006, American Institute of Physics).

Nanoscale, 2009, 1, 40–49 | 45

potentiostatic electropolymerization were performed together

with SPR to trigger the electrochemical reaction and monitor

the oxidation current involved in polymer formation and

deposition (Fig. 8, right).

AFM + ellipsometry

Although not sharing all features of conventional ellipsometry,

scanning near-field ellipsometric microscopy (SNEM) was first

introduced in 2001 as a combination of an AFM and an ellips-

ometer-like setup in an attempt to characterize simultaneously

the optical and non-optical properties of surfaces.20 The instru-

mental setup greatly resembles the AFM + SPR configuration

though in this case, a polarizer, a compensator, and an analyzer

are inserted in the incident and reflected light paths, respectively

(Fig. 9, left).

Ellipsometry detects the changes of polarization of incident

light when it is reflected from a surface. The polarization change

is expressed in the so-called ellipsometric angles, which are

related to the optical properties of a thin film, i.e. thickness

and refractive index, through the fundamental equation of

ellipsometry.

r ¼ rp

rs

¼ tanðJÞeiD

Where rp and rs are the reflectivity coefficients for the parallel and

perpendicular components of the reflected light. The ellipso-

metric angles J and D can be obtained, among other means,

from the configuration of the analyzer and the compensator

for which no reflected light is detected. This is called null

ellipsometry.

Using metal-coated tips for the AFM and configuring the

optical setup in order to produce total internal reflection on the

sample backside surface, topology and SNEM images of thin

polymer films were simultaneously obtained (Fig. 9, right).

SNEM actually maps reflected light intensities from the sample

backside surface rather than ellipsometric angles, and thus the

origin of the optical contrast in the SNEM images is, to date, not

fully understood.20

Fig. 9 Left: Instrumental configuration for AFM + ellipsometry. Null ellipso

an analyser (A) in the detector arm. Right: AFM and SNEM images of a poly

0–265 mV (b), 0–1.14 mm (c) and 0–42 mV in d) (reprinted with permission f

46 | Nanoscale, 2009, 1, 40–49

AFM + QCM

The first attempt to implement the combination of these two

non-optical techniques was reported in 1998.18 Quartz crystal

microgravimetry relies on the vibration of a piezoelectric device

(usually an AT-cut quartz slide) to detect and quantify mass

deposition and changes in viscoelastic properties. The quartz

crystal microbalance device can be made to oscillate by the

application of an alternating electrical potential (i.e., piezodriving

potential), to electrodes on the top and bottom surface of the

device, producing a shear displacement on the exposed surfaces

of the electrodes. Variations in the vibration frequency of the

quartz device will occur as a consequence of either mass depo-

sition or depletion upon the sensing area (usually one of the

electrode surfaces), while damping in the oscillation is typically

associated with material softness and/or interaction with

a viscous medium, such as an aqueous solution. In particular,

adsorbates on the device surface will interact with the

surrounding viscous medium in a manner that is dependent on

the particle size and shape. Connection of the QCM response to

changes in the shape of the adsorbate or surface coverage is not

straightforward. Modelling of QCM data is required, which in

some cases (i.e., discontinuous adsorbate layers) is either

complicated or not available.

Combining the two techniques in one instrument is thus highly

convenient for the interpretation of QCM data. The setup

demands the simultaneous use of the QCM sensing electrode as

substrate for AFM investigations (Fig. 10a). Care must be taken

in the application of oscillation amplitudes, which should be

below the required lateral resolution for AFM experiments.13

The coupled techniques have been applied to the study of ferritin

adsorption, a quasi-spherical protein that has been shown to

undergo a small deformation when adsorbed on gold. This

conclusion is sustained through comparison of surface coverages

obtained from AFM data, with QCM and finite element simu-

lation data.19

One step beyond in instrumentation involves the three-in-one

versions, where the sensing substrates are also employed as

working electrodes to perform in situ electrochemistry4,13,18

metry requires a polarizer (P) and a compensator (C) in the laser arm and

crystalline film of a thermotropic liquid crystal (gray scales 0–1.12 mm (a),

rom ref. 20. Copyright 2001, American Institute of Physics).


Fig. 10 Scheme of the experimental setup. (a) Two-in-one: combined AFM and QCM; (b) Three-in-one: combined AFM, QCM and electrochemistry

(also called electrochemical QCM or EQCM). The setup is compatible with either tip-moving AFM (tip on the scanner) or sample-moving AFM (sample

on the scanner). RE refers to reference electrode, CE to counter electrode and WE to working electrode (substrate). Inset in (b): top-view of the substrate,

showing the configuration and geometry of the electrodes.

(Fig. 10b). Again, the simultaneous application of electro-

chemical (approx. 0.03 Hz) and piezodriving potentials (the latter

about 4.7 MHz) is possible since both electrical signals widely

differ in frequency. This modality has been proved to be

particularly useful in studying the electrodeposition of metals on

platinum and gold electrodes by cyclic voltammetry,4,14,18 where

the ratio of frequency to damping shifts could be attributed to

differences in surface roughness as a consequence of metal

deposition (Fig. 11). In particular for copper electrodeposition,

the mass of trapped liquid has been estimated.4

AFM + Kelvin method: Kelvin probe force microscopy

Making probes conductive opens up the possibility of combining

electrical and scanning probe techniques. In particular, Kelvin

probe force microscopy, KPFM, combines the Kelvin method

and the atomic force microscope to obtain, together with surface

topography, the distribution of the contact potential difference

(CPD) at nanoscale resolution.

The CPD, VCPD, is the difference of the work functions of two

conductive materials, which are brought into contact. The

magnitude varies with temperature, but also with the presence of

a third material between the contacts, such as an oxide layer,

embedded dopants or adsorbed molecules.

To measure the CPD an oscillatory and a bucking bias voltage

are applied between the probe, a gold-,32,35 chromium/plat-

inum-24 or a tungsten-coated tip,40 and the substrate, forming

a capacitor. The ac voltage frequency, uel, typically differs from

that of the piezoinduced vibration of the cantilever for a better

detection of both signals. As a consequence, the cantilever starts

to oscillate. The resulting force has three different contributions,

a bias and two oscillatory terms at frequencies of uel and 2uel,

respectively, as the formula shows

Fðz;tÞ ¼�1

2

dC

dzV 2¼FdcðzÞþFðz;ueltÞþFðz;2ueltÞ¼

¼� dC

dz

�1

2ðVdc�VCPDÞ2þ

1

4V 2

ac�ðVdc�VCPDÞVacsinðueltÞ

þ 1

4V 2

accosð2ueltÞ�


Using lock-in techniques, it is possible to detect the signal at

a particular frequency, i.e. F(z, uelt). VCPD is thus obtained

by proper tuning of the bias voltage, Vdc, that nulls the

sinusoidal force signal at uel (Vdc ¼ VCPD) (Fig. 12). Alter-

natively to force nulling, other methodologies detect and

cancel the Kelvin current across the tip and substrate by the

bias voltage.6

Developed as a combined technique in 199132 KPFM has

rapidly improved in versatility and lateral resolution. The tech-

nique was initially applied to the characterisation of metal

surfaces32 at low, micrometric resolution. The application to

organic films came shortly after, with the first characterizations

of films of mixed amphiphilic molecules,8 self-assembled mono-

layers26 and a biological membrane.21 Recent years have wit-

nessed the use of KPFM in characterising chemically

lithographed self-assembled monolayers35 and the substantial

increase of its lateral resolution to the submicron range. Exam-

ples of that are contact potential maps of films of pulmonary

surfactants24 or gap structures in microelectrodes10 (Fig. 12).

New applications of KPFM in biotechnology have abounded

and developed in parallel. In these cases, KPFM is often pre-

sented as a non-destructive, label-free method for the localization

and detection of proteins and nucleic acids and thus as an

alternative technique to the classical fluorescence and mass

spectrometric methods in proteomics.23,40 Cell membrane char-

acterization of living cells7 and single-molecule detection25 are

among its most recent applications.

Conclusions

The last decade has witnessed the development of combined

AFM-based techniques aimed at a more complete description

of processes and structures at the nanoscale. The technical

effort has been considerable, and, especially in the case of

tip-enhanced techniques or the fruitful partnership with

optical microscopies, the applications have been numerous

and of invaluable relevance, particularly in the field of biosci-

ences. However, the applications based on combinations of

AFM with classical surface techniques such as QCM and

Nanoscale, 2009, 1, 40–49 | 47

Fig. 12 Left: Scheme of the basic principle of KPFM. A sinusoidal [Vac sin(uelt)] and a bias voltage are applied across the sample. As a result, the tip

oscillates at uel and at 2uel. VCPD is obtained by nulling the signal at uel. Right: AFM image (A) and KPFM image (B) of a gap between 4 nm-thick

platinum (Pd) and titanium (Ti) electrodes (substrate, SiO2/Si). (C) is a profile of the discontinuous line across the metal gap depicted in (B), showing the

lateral resolution of the KPFM image as the lateral distance between the contact potentials of Pd and Ti (approximately 100 nm, reproduced with

permission from ref. 10. Copyright 2007. American Chemical Society).

Fig. 11 Left column: Silver deposition on gold. Right column: Copper deposition on gold. Top image: AFM topography image showing the effects of

metal deposition. Evolution of the electrochemical potential (first plot), 3rd overtone frequency and damping shifts measured with QCM (second and

third plots, respectively), and current and time derivative of the frequency shift. For copper, bigger granules are deposited and the higher roughness

detected with AFM may help to interpret the higher damping values obtained for this system as compared to those obtained for silver. (Reproduced with

permission of ECS – The Electrochemical Society from ref. 13).

ellipsometry have been rather scarce. Their potential is still

intact though, since data interpretation remains a pending

issue. Kelvin probe microscopy, still waiting for further

development, open new ways to look at single molecules,

membranes and cells.

48 | Nanoscale, 2009, 1, 40–49

Acknowledgements

The authors thank the Etortek Program of the Basque Govern-

ment (IE07/201) and the National Plan project (CTQ2007-

66541). Uwe Rietzler and Rafael Benitez are acknowledged for


providing literature references and Kathryn Melzak for

grammar/spelling correction and expert advice on SPR

and QCM. JLTH thanks the I3 programme of the Spanish

Government.

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The new future of scanning probe microscopy: Combining atomic force microscopy with other surface-sensitive techniques, optical microscopy and fluorescence techniques

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