Review

Strategies and results of atomic force microscopy in the study

of cellular adhesion

Anne Simon*, Marie-Christine Durrieu

INSERM U577 ‘Biomaterials and tissue repair’, Universite Victor Segalen Bordeaux 2, 33 076 Bordeaux, France

Received 10 February 2005; revised 21 June 2005; accepted 22 June 2005

Abstract

Atomic Force Microscopy (AFM) provides a range of strategies for investigating living cell adhesion to the extracellular matrix, other cells

or biomaterials in their native environment. This review surveys the results obtained from major studies using AFM for mechanical force

evaluation in the cell, morphological visualization of the cell and studies of the cell’s response to chemical or mechanical stress. Recently, the

use of AFM has been broadened to obtain experimental information about cell adhesion molecules. Quantitative measurements of binding

forces between adhesion proteins and their ligands in the cell or on a surface are presented. These analyses provide data on individual

molecules and their resulting collective behaviour at the cell level. They significantly contribute to the characterisation of cellular adhesion

with physical principles relating to biochemistry.

q 2005 Elsevier Ltd. All rights reserved.

Keywords: Atomic force microscopy; Force spectroscopy; Cell adhesion; Cell surface; Cell cytoskeleton; Cell elasticity; Cell–cell interaction

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

2. Principles of an AFM experiment on cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

3. Mechanical properties of cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

3.1. Image processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

3.2. Force curve analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

3.3. Force mapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

4. Cell adhesion to surface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

4.1. Morphologic evaluation of cell adhesion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

4.2. Adhesion strength measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

5. Cell-to-cell adhesion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

5.1. Interaction forces between cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

5.2. Binding strength between individual cell adhesion molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

0968-4328/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.micron.2005.06.006

* Corresponding author. Tel.: C33 5 57571115; fax: C33 5 56900517.

E-mail address: abjsimon@hotmail.com (A. Simon).

1. Introduction

The attachment of cells to other cells or to an extra-

cellular matrix plays a key role in many biological (Basson

et al., 1992; Bourdoulous et al., 1998; Folkman and

Moscona, 1978; Ingber, 1990; Stossel, 1993) and patho-

logical (Klotz, 1992; Pauli et al., 1990) processes such as

embryogenesis, mitosis, leukocyte traffic, motility or

Micron 37 (2006) 1–13

www.elsevier.com/locate/micron

A. Simon, M.-C. Durrieu / Micron 37 (2006) 1–132

metastasis formation in tumor-bearing patients and throm-

bus formation. In vitro, most cells are anchorage-dependent

and attach firmly to the substrate in order to divide

(Folkman and Moscona, 1978).

The cell’s capacity to make specific contact is mediated

by adhesion proteins (Bongrand, 1998); such as cadherin or

integrin (Kreis and Vale, 1999; Springer and Wang, 2004).

These protein receptors elicit a cellular response upon

ligand binding. Intracellular signalling leads to stabilization

of the contact cell or to cell motility (Hynes, 1992; Ruoslahti

and Pierschbacher, 1987).

Since cellular adhesion involves collective behaviour of

individual proteins, this process is difficult to study.

Experimentally, approaches have been developed to

characterize the process according to cell mechanical

properties as well as cell morphology, for example, and to

study the modulation of the cell properties consistent with

the adhesion receptor attachment to a surface or cytoskeletal

organization.

Quantitative information on the adhesion strength and

mechanical parameters of living cells can be obtain by

applying external forces. With shear flow assays, adherent

cells on a flat surface are submitted to a laminar flow;

weakly attached cells are deformed, and the cell-substrate

contacts are disrupted: cells are carried away and the

remaining cells are counted (Chan et al., 1992). A model

explains the dissipation of the mechanical energy due to the

work of the applied forces. Centrifugal assays may also be

used. They have recently been optimised to better define the

force applied to cells (McClay et al., 1981; Thoumine et al.,

1996). Mechanical properties of cells have also been probed

by other experimental approaches such as micropipettes

(Discher et al., 1994; Evans, 1989), traction forces on cells

(Galbraith and Sheetz, 1997; McClay et al., 1981), new

techniques such as optical tweezers (Ashkin and Dziedzic,

1989; Florin et al., 1997), magnetic tweezers (Bausch et al.,

1998, 1999).

Cell morphology (cell shape and spreading) can be

visualized by microscopy and it is also an initial indication

of adherent cell behaviors. Standard light microscopy is a

straightforward non-invasive method, which can distinguish

distinct morphology classes and state of adhesion: spheroid

cells for a non-adherent cell or spreading cells for an

adherent cell. Massia and Hubbell (1992) also reported SEM

images of fixed cells, and categorized the morphologies of

fibroblast cells as either spheroid cells with zero, one, two,

or more than two filipodia extensions or as flattened cells.

Non-adherent cells are expected to be spheroid with no

filipodia extension. Finally, techniques such as immuno-

fluorescence staining microscopy are capable of identifying

intracellular structures such as cytoskeleton fibers presented

by adherent cells (Charras and Horton, 2002a,b).

Atomic Force Microscopy (AFM) (Binnig et al., 1986) is

emerging as a valuable tool for studying cellular adhesion,

because of its ability to maintain cell viability, so that living

cells may be observed in their culture medium without

inducing irreversible damage (Hansma and Hoh, 1994;

Henderson, 1994; Kasas et al., 1997). AFM results combine

image information about cell morphology with the

mechanical properties of the cell surface such as cellular

elasticity with 2D lateral resolution or measure cell–surface

or cell–cell interaction forces.

The aim of this review is to present the experimental

results of cellular adhesion studies using AFM. On one

hand, AFM allows quantification of mechanical properties.

On the other hand, AFM may be used not only to examine

the cell morphology, but also to mechanically stress the cell

and then, observe the cell’s response to the disturbance and

its influence on cell attachment. Finally, we will review

results from studies on cell-surfaces or cell-types following

the pioneering studies of Sagvolden et al. and Thie et al.

(Sagvolden et al., 1999; Thie and Denker, 1997; Thie et al.,

1998) or the experimental study of ligand–receptor

interaction at the single-molecule level.

2. Principles of an AFM experiment on cells

The principle of AFM will only be briefly described here

as it is presented in depth in many books and review articles

(Binnig et al., 1986; Dufrene, 2003; Henderson, 1994; Hoh

and Hansma, 1992; Jena and Horber, 2002). For further

details, interested readers may refer to them.

The basis of AFM consists of scanning a surface with a

very sharp tip mounted on a cantilever (Fig. 1). The

deformation induced on the tip is recorded with an optical

detection system (Alexander et al., 1989; Meyer and Amer,

1988); and this deformation may be directly translated into a

force with picoNewton accuracy. A piezoelectric scanner

controls motion of the surface with sub-Amstrong precision.

Topographic information about the surface can be

obtained in two modes: intermittent contact and contact

(Fig. 2). In the intermittent contact mode (Nagao and

Dvorak, 1999; Tamayo et al., 2001; Vie et al., 2000), the

cantilever is vibrated on the sample. Because the tip is in

intermittent contact with the sample surface, the frictional

force and the contact time can be reduced and it can be used

to image fragile or loosely attached samples (Hansma et al.,

1994; Putman et al., 1994). Some studies have examined the

topography of unfixed cells using this approach (Butt et al.,

1990; Horber et al., 1992; Le Grimellec et al., 1994, 1998).

In the contact mode, the tip is brought into continuous

contact as it scans the surface. The tip–sample interaction is

kept constant throughout the imaging process. Since the

biological sample observed is not flat and since the tip can

interact differently with different areas on the sample, the

vertical position of the sample varies in order to maintain

interaction while the tip is scanning the specimen. To

prevent the tip from moving the cells, cells can be trapped in

pores of filters (Kasas and Ikai, 1995). They may

spontaneously adhere to the surface of a glass coverslip. It

is also possible to coat a glass or a silica wafer with

Fig. 1. AFM tip geometries. (A) Pyramidal cantilever (bar is 10 mm) and small tip (inset, bar is 1 mm). (Kumar and Hoh, 2001, Probing the machinery

trafficking with the Atomic Force Microscope, Traffic, q 2001 Blackwell Publishing) (B) Microbead glued to the cantilever. (C) Coated with human

trophoblast-type cells. Scale bar is 5 mm. (Thie et al., 1998).

Fig. 2. AFM imaging modes. (A) Intermittent contact mode. (B) Contact

mode (Kumar and Hoh, 2001, Probing the machinery trafficking with the

Atomic Force Microscope, Traffic, q 2001 Blackwell Publishing).

A. Simon, M.-C. Durrieu / Micron 37 (2006) 1–13 3

positively charged material such as poly-lysine, or adhesive

proteins in order to improve the adhesion of the cells to a flat

surface (Altankov and Groth, 1994; Liu et al., 2003; Puleo

and Nanci, 1999; Ruardy et al., 1995). Prior to cell imaging,

the scanning force is adjusted with force curves. The vertical

force applied is adjusted to a minimum force. Typically, the

force applied is situated between 20 pN and 1 nN (Le

Grimellec et al., 1998). It is set in the force calibration mode

and collected by measuring the cantilever deflection as the

sample is moved toward the tip. Note that AFM is

minimally disruptive to cells using a standard AFM tip

(Parpura et al., 1995) or when used for short periods of time,

around a few hours (You et al., 2000). Many cell types such

as living neurons and glia (Parpura et al., 1993), endothelial

cells (Mathur et al., 2000, 2001), epithelial cells (Hassan et

al., 1998; Henderson and Oberleithner, 2000; Hoh and

Schoenenberger, 1994), and fibroblasts (Rotsch and

Radmacher, 2000) have been imaged using AFM. Nuclei,

mitochondria and cytoskeletal filaments have been

identified.

A. Simon, M.-C. Durrieu / Micron 37 (2006) 1–134

AFM can be carried out either in air or in near-

physiological liquid conditions (Drake et al., 1989; Gould

et al., 1990). Better resolution is obtained in air, on fixed

cells (Hoh and Schoenenberger, 1994), but dynamic cell

behavior and the mechanical features of the cells are not

accessible.

The results obtained by AFM in studies of living cells let

expected, in future work, an improvement in time

resolution. In order to follow processes in situ, with AFM,

the data acquisition time is limited as it takes a few minutes

to capture an image, whereas many biological and

biochemical processes occur in less than a minute. Faster

scanning rates should be developed in the future.

3. Mechanical properties of cells

Cell mechanical properties are controlled mainly by the

cytoskeleton and by membrane elasticity (Sackmann, 1994).

As intracellular cytoskeleton organization is connected to

the surface by adhesion receptors, it is associated with

cellular adhesion. The atomic force microscope has

emerged as a powerful apparatus for obtaining mechanical

information about the sample under observation. The

mechanical properties of the sample are obtained by

image processing, by force curve analysis or by force

modulation methods. We shall now review these tree

methodologies.

3.1. Image processing

One method consists in recording height images at a

constant load. These combine topographical and mechanical

Fig. 3. Cytoskeletal fiber height by plotting a cell profile and AFM images. I

properties of the imaged cell. From the height profile

(Fig. 3) of the scanned cell, one can extract the tip

indentation depth at different locations on the cell from

which the elastic modulus G is deduced:

F Z kd Z PGf=ð1Kn2Þ (1)

The variation of the elastic modulus G as a function of

the indentation d is due in part to the cell heterogeneity

and to the finite size of the tip. It is a suitable way to

establish a correlation between the cell’s compliance and

its local structure. In an attempt to model of cell local

mechanical properties deduced from the analysis of

height images (Simon et al., 2004), a weak adherent

cell appeared to exhibit a more homogeneous elastic

response than a cell adhering firmly to the surface, as

expected.

3.2. Force curve analysis

The force curves give the variation of the cantilever

deflection, dc, as a function of the imposed vertical

displacement of the sample, D. They can be fitted with the

relation dcZSD, where dc is the cantilever deflection, D is

the ceramic displacement, and S is the slope of the force

curve containing the mechanical properties of the cell

studied.

To extract an elastic modulus from the curve, a model, as

specified in several references (Domke et al., 2000;

Radmacher, 1997; Simon et al., 2003; Vinckier and

Semenza, 1998), and based on the theory of Hertz and of

Sneddon is used to describe the elastic indentation of the cell

surface by the tip (Hertz, 1882; Sneddon, 1965).The local

stiffness ks of the cell surface is given by: ksZGf, where G

mage scan size: 130!130 mm. Z-scale: 0–15 mm (Simon et al., 2003).

A. Simon, M.-C. Durrieu / Micron 37 (2006) 1–13 5

and f are the elastic modulus and the diameter of the contact

area, respectively. The key point is determining the

geometric factor that shows the relation between the

indentation depths, d, and the diameter of the contact

area, f. The geometry of the tip is widely considered to

resemble a cone shape (Hertz, 1882; Sneddon, 1965).

This approach has been widely used in similar situations

(Boschung et al., 1994; Domke and Radmacher, 1998;

Domke et al., 2000; Haga et al., 2000; Mathur et al., 2000;

Rotsch and Radmacher, 2000).

With the help of two equations, one corresponding to the

equality of the force between the elastic medium and the

cantilever, kc dcZks ds and the other giving the vertical

displacement as the sum of the cantilever deflection and

indentation depth, DZdcCds one can immediately calculate

the expression of the slope, S, of the force curve (the plot of

dc versus D) that contains information on the elastic

properties (Fig. 4).

Many experimental reports evaluate the elastic properties

of different cells. Mathur et al. (2001) determined the

mechanical properties of three different cell types. They

Fig. 4. (A) Forces curves obtained on the hard surface and on a soft material, su

surface (symbols) and theoretical calculations (line) (Simon et al., 2003). (C) T

Reprinted from Colloids and Surfaces B: Biointerfaces, 19, Domke et al., Substra

investigated by atomic froce microscopy, 367–379. q 2000 with permission from

showed that cardiac cells were the stiffest (100 kPa),

skeletal muscle cells were intermediate (25 kPa) and

endothelial cells were the softest (1.4–7 kPa) depending

on the location of the cell surface tested, consistent with the

function of these cells. As reviewed in this article (Fig. 5), a

wide range of values of elastic modulus (0.6 kPa–0.67 MPa)

can be obtained, depending on the cell types and on the

location of the cell surface.

By combining AFM and Total Internal Reflection

Fluorescence Microscopy (TIRFM), the mechanical force

transmission measurements were simultaneously examined

with focal contact dynamics in cultured cells monolayer

(Mathur et al., 2000). In this way, the study provided

evidence of force transmission from the apical cell

membrane to the basal cell membrane globally, resulting

in rearrangement of contacts on the basal surface.

3.3. Force mapping

The force mapping mode has been used to access local

structure information. The sample is scanned laterally,

rface cell. (B) Forces curves obtained on different zones of the cell-bound

wo-dimensional map of the elasticity of the sample (Domke et al., 2000.

te dependent differences in morphology and elasticity of living ostecblasts

Elsevier).

Fig. 5. Cell mechanical properties measured by AFM. Literature (Mathur et al., 2001. Reprinted from Journal of Biomechanics, Endothelial, cardiac muscle

and skeletal muscle exhibit different viscous and elastic properties as determined by atomic force microscopy, 1545–1553. q 2001, with permission from

Elsevier).

A. Simon, M.-C. Durrieu / Micron 37 (2006) 1–136

and force curves are taken at each point. The force curves

are analysed to calculate a quantitative value for the local

elastic modulus at each location on the cell. Due to

hydrodynamic interaction and the travel scale, the scan

rate is limited and fast cellular processes cannot be tracked

in this way; and it is useful in this case, to process images.

Nevertheless, the result is a two-dimensional map of the

elasticity of the sample, as shown in Fig. 4C (Domke et al.,

2000). The substrate appears stiffer than the soft part of the

cell. The cytoskeletal fibers present increase elastic modulus

as compared with the body of the cell. Rotsch and

Radmacher (2000) affected the integrity of different

components of the cytoskeleton using drugs and measured

the elastic modulus of the cell. They showed the importance

of the actin network compared to the microtubule network

on the cell’s elastic properties and on the mechanical

stability of living cells.

4. Cell adhesion to surface

4.1. Morphologic evaluation of cell adhesion

AFM is a powerful experimental approach for the

morphological characterization of the cell adhesion. The

morphologic evaluation of bone cells by AFM will be taken

as an illustration, since understanding the interaction of

bone cells with implants is of obvious interest for

manufacturing bone materials (Puleo and Nanci, 1999).

The biocompatibility of materials is also related to the way

cells behave in contact with them.

The study of the adhesion of bone cells with different

materials has been performed using AFM by several teams

(Charras and Horton, 2002a,b; Domke et al., 2000;

Lehenkari and Horton, 1999; Simon et al., 2003). These

studies have provided an initial characterization of cell

adhesion by AFM images which show different cell shapes

and flattening: cell spreading could be distinguished

(Fig. 6A). The shape of the cells can also be defined by

parameters such as cell area, cell height or the flatness shape

factor, as shown by Domke et al. (2000). As in other studies,

it is assumed that cell shape gives a strong indication of the

quality of adhesion (van Wachem et al., 1989). Moreover, it

is accepted that flatness is directly correlated with good

adhesion, while this is not the case for more compact and

higher cells. Finally, the presence of an extensive network

of fibers inside the cell which constitutes part of the

cytoskeletal structure indicated, in addition to the shape and

the spreading of the cells, direct establishment of cell

adhesion (Simon et al., 2003).

These characterizations are consistent with those

obtained by SEM (Simon et al., 2003) and by immuno-

fluorescence staining microscopy which confirm the nature

of the fibers observed on AFM images since F-actin was

stained in this experiment (Fig. 6B; Charras and Horton,

2002a,b).

Mechanical load as well as drugs (Rotsch and

Radmacher, 2000) are also known to influence the

organization of the cell cytoskeleton bundles. Therefore,

to evaluate effective cellular adhesion, a large external force

can be applied with the AFM tip in situ (Fig. 7). When a cell

was stable and really adherent to a surface, exerting a force

on the cell did not disturb it (Simon et al., 2003). The

difference in cellular adhesion could explain the effect of the

different loads exerted by the tip on the cell.

Morphological characterization of cellular adhesion may

be revealed by AFM at different levels, first distinguished by

the presence of developed and structured stress fibers and by

the cell’s stability when the cell undergoes an applied load

or the effect of drugs.

Fig. 6. (A) Morphological aspect of osteoblast spreading observed by contact-mode AFM. Image scan size: 130!130 mm. Z-scale: 0–15 mm (Simon et al.,

2003). (B) Characterization of cytoskeleton actin fibers and focal contact protein paxillin, observed by immunofluorescence staining microscopy. Scale bar is

10 mm (Reprinted from Biophysical Journal, Charras and Horton, 2002a, with permission from the Biophysical society).

A. Simon, M.-C. Durrieu / Micron 37 (2006) 1–13 7

4.2. Adhesion strength measurements

Sagvolden et al. (1999) reported a very interesting AFM

study of the quantification of cell adhesion forces. They

adapted AFM and introduced a manipulation force

microscope. The principle is shown on Fig. 8. An inclined

Fig. 7. Detachment of osteoblasts observed by contact-mode AFM. Imag

AFM cantilever is used like a piston fitted with a spring, to

laterally dislodge a cell. Before displacement, the inclined

cantilever was aligned with the cell using an inverted optical

microscope. No force was recorded after all cell-substrate

bonds had been removed. This apparatus measures the force

necessary to displace an object adhering to a substrate.

e scan size: 130!130 mm. Z-scale: 0–15 mm. (Simon et al., 2003).

Fig. 8. AFM adapted and cell displacement (cartoon of the cell displacement, cell as viewed in the optical microscope and the applied force versus

displacement) (Sagvolden et al., 1999; q 1999 National Academy of Sciences, U.S.A.).

A. Simon, M.-C. Durrieu / Micron 37 (2006) 1–138

The force measurement, when displacing a cell adhering

to a substrate, is obtained by recording the laser beam

deflection versus the lateral cantilever displacement.

A force–distance curve is obtained. The baseline to peak

force DF, the peak width Dx and the work DW, were

calculated from the data. As emphasized by the authors,

the measured force depends on the direction and the speed at

which the cell is displaced. This approach also estimates the

time before adhesion forces are observed and the time over

which adhesion saturates (Fig. 9). Cervical carcinoma cells

attached faster and stronger at 37 8C than at 23 8C and better

on a hydrophilic than on a hydrophobic surface. The force

was of the order of 100–200 nN, depending on the coating

substrate (Fig. 9). As studied by Yamamoto et al. (1998), the

force between 300 and 400 nN was necessary to dislodge

murine fibroblast cells. In this case, the technique used was

similar to the manipulation force microscope. Interestingly,

these observed forces are similar to those obtained by other

techniques. By high-speed centrifugation, the force required

to dissociate epithelial cells from the surface was 100 nN

(Thoumine et al., 1996). The measurements with an adapted

AFM give detailed information on the adhesion forces as

a function of time, temperature and the hydrophobicity

of the substrates.

5. Cell-to-cell adhesion

5.1. Interaction forces between cells

Force spectroscopy investigates cell-to-cell interaction

down to the molecular level. This approach has emerged

Fig. 9. Cell-substrate adhesion forces on cell-culture surfaces. Median forces as a function of time for cells seeded on hydrophilic (Phil) polystyrene coated with

bovine serum albumin (BSA), fibronectin, or laminin. Table of cell adhesion measurements from Sagvolden et al. (1999) work q 1999 National Academy of

Sciences, U.S.A.

A. Simon, M.-C. Durrieu / Micron 37 (2006) 1–13 9

recently to measure the behaviour of a molecule or a cell

under stretching mechanical force. As reviewed earlier

(Fotiadis et al., 2002), single biomolecules can be imaged,

manipulated and dissected in a controlled manner, and

inter- and intra molecular forces can be measured (Benoit

and Gaub, 2002; Hinterdorfer et al., 1996; Lehenkari and

Horton, 1999). Although much work has been done to

quantify the binding forces of interactions between protein

receptors and their ligand plated on a surface or on cell using

AFM, it is not easy to provide quantitative data on specific

interaction forces between cell layers. An ideal experiment

has done by Thie et al. (1998) and consisted of (1) bringing

cells into contact with monolayers of cells at know

indentation threshold force, (2) waiting for some time, and

then, (3) separating them. Repulsive forces exerted between

cells could be determined in step 1. In the next step, after the

initial contact between cells adhesive interactions develop

slowly, and are measured in step 3. This procedure of n

‘approach and separation’ cycles could be repeated a

sufficient number of times to obtain reproducible results

because of the complexity of living cells. These series of

experiments could also be repeated for different values of

contact times.

In this study, Thie et al. used this approach to study cell–

cell adhesion (Thie and Denker, 1997; Thie et al., 1998). As

a force-sensor, a microbead is mounted on cantilevers

(Johnsson et al., 1991) (Fig. 1b). This AFM sensor was

functionalized with human trophoblast-type cells (JAR)

(Fig. 1c). JAR-coated force sensors were brought into

contact with confluent monolayers of human uterine

epithelial cells (HEC-1-A, RL95-2), for different periods

of time (Fig. 10).

The approach force curves displayed repulsive inter-

actions for both HEC-1-A and RL95-2 cells. But RL95-2

cells showed lower values for the repulsive regime than

HEC-1-A cells. This repulsion is attributed by the authors to

the peripheral structure of the cell (e.g. glycocalyx). They

have previously shown that HEC-1-A cells posses a thicker

glycocalix than RL95-2 cells (Thie and Denker, 1997; Thie

et al., 1998). After having overcome repulsive interactions,

the initial contact was followed by adhesive interactions

(Fig. 10). The adhesion increased with increasing contact

time. For prolonged contact times (20 and 40 min), both cell

types showed a marked difference in the cell separation

force curves; particularly after the maximum adhesion

force. The force between JAR and HEC decreases

continuously in small steps of !200 pN upon separation,

while RL showed specific JAR binding with repeated

rupture events in the range of 1–3 nN, attributed to the

formation of strong cell-to-cell bonds.

This force spectroscopy on live cells was applied to a

variety of different cell adhesion systems such as bacterial

adhesion (Razatos et al., 1998), and leukocyte–endothelial

interaction (Zhang et al., 2004).

5.2. Binding strength between individual cell adhesion

molecules

A pioneering study was performed by Dammer et al.

measuring the binding strength of proteoglycan to

Fig. 10. (A). Force–distance plot and schematic illustration of the experiment (reprinted from Methods in Cell Biology, 68, Jena and Horbes, AFM in cell

biology, 91–114, q 2002, with permission from Elsevier). (B) Typical adhesive forces curves for HEC and RLG cells resulting when a bovine serum albumin-

coated microbead or a JAR-coated microbead (Thie et al., 1998). (Reprinted from Methods in Cell Biology, 68, Jena and Horbes, AFM in cell biology 91–114,

q 2002, with permission from Elsevier.)

A. Simon, M.-C. Durrieu / Micron 37 (2006) 1–1310

proteoglycan interactions (Dammer et al., 1995; Popescu

and Misevic, 1997). Glyconectin 1 proteoglycans purified

from marine sponge species were covalently cross-linked to

the cantilever and to a flat mica surface. Like cell-to-cell

interaction force spectroscopy, the approach–retract cycles

were repeated and an intermolecular force of about of

400 pN resulting from about 10 individual interactions of

40 pN each was found. Moreover, glyconectin 1–glyconec-

tin 1 interactions were inhibited with block 2 anti-

glyconectin 1 monoclonal antibodies (which were shown

to completely block glyconectin promoted cell and bead

adhesion). It clearly established that these proteoglycan–

proteoglycan interactions mediate cell adhesion and

produce fundamental cell cohesion-forces in the sponge,

like protein-carbohydrate binding immunoglobulin, cad-

herin, lectin and extracellular matrix families of cell

adhesion molecules. In another study, Oberhauser et al.

(1998) studied the mechanical properties of the extracellular

A. Simon, M.-C. Durrieu / Micron 37 (2006) 1–13 11

matrix protein tenascin, which mediates the attachment and

the rolling of cells in shear flow. They showed the dynamic

extensibility of tenascin: single molecules of tenascin could

be stretched to several times its resting length. This property

could allow the tenascin-receptor bond to persist over long

extensions. Similar results were obtained in studies of

the giant muscle protein titin (Rief et al., 1997). Authors

showed that the elasticity of titin is determined by

the composition of elastic Ig domains within the protein

structure which in turn determine the macroscopic

mechanical behavior of this protein.

In a very interesting study, Marshall et al. (2003)

compared lifetime measurements from AFM and flow

chamber assays. They reported that at low forces

(!50 pN), the selectin-PSGL1 (P-selectin glycoprotein

ligand 1) bond lifetime increases with force, whereas at

high forces (O50 pN), it decreases with force. They show

that the mechanical response of the selectin-PSGL1 bond

depends on the force history.

These quantitative measurement examples on the single

molecule level investigate fundamental principles of

adhesion proteins structure and function.

6. Conclusion

This review has dealt with cellular adhesion studies using

AFM. With this technique living cells can be investigated in

their physiological environment through the examination of

the mechanical properties as well as the morphology of the

cells. AFM allows the exploration and the measurement of

mechanical properties of cells related to the local cell

structure. Elastic modulus can be collected. It is possible to

image the adhesion state of cells under native condition and

also to follow cellular processes such as deadhesion by

applying a mechanical load with the AFM tip or by injecting

drugs. An investigation of different factors (temperature,

substrate, etc.) involved in cellular adhesion can be

evaluated by a direct measurement of the force necessary

to detach a cell from a surface. AFM must be adapted for

these measurements, by inclining the cantilever. Finally,

AFM provides direct measurements of cell-to-cell inter-

action down to the molecular level. In the future, these

experimental developments will find applications in

investigations of mutated cell adhesion proteins or the

coupling of cellular adhesion molecule with the cytoskele-

ton on the affinity of cell for materials or other cells.

Acknowledgements

The authors would like to thank Dr A. Fuchs for helpful

discussions and for critical reading of the manuscript.

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