Figure 2
Schematic of the major components of an atomic force microscope,
showing the feedback loop for TappingMode operation.

AFM History and Methods
AFM is the most commonly used
form of the scanning probe
microscopy (SPM) family of techniques. The origin of SPM beganwith the development of the scanning tunneling microscope (STM) in
1982 by researchers at IBM,
Zurich.9 The ability of the STM toresolve atomic structure on a sample surface earned the inventors theNobel Prize in 1986.
However, the STM can only beapplied to conductive or semiconductive specimens. To broaden thistype of microscopy to the study ofinsulators, the atomic force microscope was developed in collaboration between IBM and Stanford
University in 1986.10 AFM is
performed by scanning a sharp tip
on the end of a flexible cantileveracross a sample surface, while
maintaining a small, constant force(see fig. 1). The tips typically havean end radius of 5 nm to 10 nm,
although this can vary dependingon tip type. The scanning motion isconducted by a piezoelectric tube
scanner that scans the tip in araster pattern with respect to thesample (see fig. 2). The tip-sample
interaction is monitored by reflecting
a laser off the back of the cantilever
onto a split-photodiode detector.
The two most commonly used
modes of operation are contact
mode AFM and TappingMode™
AFM, which are conducted in airand liquid environments.
In contact mode AFM, a constantcantilever deflection is maintainedby a feedback loop that moves thescanner vertically (z) at each lateral(x,y) data point to form the topographic image. By maintaining a
constant deflection during scanning,a constant vertical force is maintained between the tip and sample.

Applied forces during imaging
typically range between 0.1 and
100 nN. Although contact mode
has proven useful for a wide range
of applications, it sometimes has
difficulty on relatively softsamples.
TappingMode AFM consists of oscillating the cantilever at its resonancefrequency (typically ~300 kHz) andscanning across the surface with aconstant, damped amplitude. The
feedback loop maintains a constantroot-mean-square (RMS) amplitude
by moving the scanner verticallyduring scanning, which correspondingly maintains a constant appliedforce to form a topographic image.
The advantage of TappingMode isthat it typically operates with alower vertical force than that possible with contact mode, and it eliminates the lateral, shear forces thatcan damage some samples. Thus,
TappingMode has become the preferred technique for imaging soft,fragile, adhesive, and particulatesurfaces.
Although the initial use of SPM wasto produce high-resolution topographic images, a number of SPM
techniques have been developed to
study physical and materials properties about sample surfaces. These
techniques are commonly used toinvestigate differences in friction,adhesion, elasticity, hardness,magnetic and electrostatic fields,carrier concentration, conductivity,and temperature distribution.
Fundamental force studies are also
conducted to study adhesive, attractive, and repulsive interactions
between specimens.
One of the important innovations
that has been integrated into commercial atomic force microscopes isenvironmental control of the sample.
Imaging at elevated temperatureshas made it possible to study
thermal phase transitions in ambient
or inert gas conditions. Imaging in
fluid at elevated temperatures has
provided the ability to image structures at a physiological temperatureof 37°C. Atmospheric hoods arealso commonly used in AFM tocontrol humidity or to conduct
experiments under specific atmospheric conditions.

Drug Interactions
Interactions between biologicalspecimens, such as ligand-receptor11 and protein-DNA12 systems,
have frequently been studied byAFM, and can also be applied tothe study of drug interactions with avariety of biological specimens. For
example, AFM-based immunological studies have investigated antibody-antigen binding interactions,13–15 and drug-DNA complexes
have been studied with AFM todetermine DNA ligand mode-ofbinding.16–19 This is of considerable
interest since nucleic acid ligands
are commonly used as anticancer
drugs and in the treatment of genetic diseases. However, determining
whether they bind to DNA by intercalation within major and minor
grooves, by “nonclassical” modes,
or by a combination of thesemodes can often be difficult andlabor intensive. AFM was used to
study drug binding mode, affinity,and exclusion number by comparing the length of DNA fragmentsthat have and have not been
exposed to the drug.17 It is known
that if intercalative binding is occurring, the DNA strand increases inlength. Furthermore, the degree oflengthening is informative in determining the binding affinity and thesite-exclusion number. AFM wasshown to be an effective means of

seeing and measuring any changes
in the DNA strand.
When exposed to ethidium, a wellcharacterized intercalator, the DNA
strand was shown through AFM tohave increased in length from3300 nm to 5250 nm (see fig. 3).
Similarly, AFM intercalative binding
studies showed the increase in theDNA strand, from 3300 nm to4670 nm, upon exposure to daunomycin, an anticancer drug used totreat leukemia. This technique has
also successfully been applied to
new drugs in which the mode ofbinding was unclear. Exposure of
2,5-bis(4-amidinophenyl) (APF), a
new drug for the treatment ofPneumocystis carinii pneumonia,
did not produce lengthening of theDNA strands, indicating that thedrug binds by nonintercalativemodes.18
Although many AFM studies haveconcentrated on interactions at themolecular level, responses of living
cells have also been visualized.
Since cells can be imaged in aphysiological solution at 37°C, it ispossible to monitor the interaction
between a metabolically active celland a chemical or biological additive to the solution environment.
Work has been published on
observing the interaction between
living cells and drugs,20,21 viruses,22–24 and other chemicals.25,26
Rotsch and Radmacher conducted“force mapping” studies on 3T3
and NRK fibroblast cells to visualize
the effects of various drugs.20 Forcemapping, also referred to as “theforce volume technique,” conducts
a lateral array of force curves
across a sample surface that results
in a force-interaction image.27
Collecting force curves consists ofrecording the change in the cantilever deflection as it moves vertically toward the sample until it
touches the surface, and then pulls
away from the surface and retracts
back to its starting position.28
Force curves can detect repulsive,
attractive, and adhesive interactionsin the piconewton-to-nanonewton
range.
In Rotsch and Radmacher’s study,
force volume was used to map cell

Figure 3
2.5 µm scans showing (left) a ligand-free DNA strand measuring ~3300 nm;(right) an ethidium-DNA complex measuring ~5250 nm. Images courtesy ofL.A. Bottomley, Georgia Institute of Technology. [Reference 17]

Figure 4
Elasticity maps showing the dissaggregation of actin filaments in 3T3 fibroblasts due to interaction with Cytochalasin
B. Images courtesy of Rotsch and Radmacher, Universität München, Germany. 230µm scan size. [Reference 20]

elasticity changes over time due tothe interaction with f-actin disabling
drugs. Actin filaments form a network that provides mechanical stability to the cell. By adding f-actin
disabling drugs (Cytochalasin Band D, Latrunculin A, and jasplakinolide) to the solution environmentof the living cells, the disaggregation of the actin filaments could bestudied directly by observing thechange in the elasticity of the cells.
In AFM, a reduced amount of cantilever deflection when the tip is incontact with the cell indicates adecrease in elasticity. In figure 4,
the effect of Cytochalasin B on theelastic modulus of the 3T3 fibroblastcan be seen over time. Before addition of the drug, the actin filaments
can be clearly seen in the force volume image (arrow in fig. 4a) due
to their high stiffness with respect tothe rest of the cell. After addition of
Cytochalasin B to the fluid chamberwith the cells, the disaggregation of
the actin network produces adecrease in elasticity, which is indicated by the darker areas in theimages. Over the 160-minute timeframe, the elasticity (Young’s modulus) was seen to decrease by a factor of 3.1. With each of the drugs

applied, the effects of drug concentration on the rate of interaction andthe decrease in elasticity weresuccessfully studied.

Disease Mechanisms
AFM is also commonly used to gaina better understanding of the physiological mechanisms associatedwith disease. Some examples ofthis include AFM studies associatedwith Alzheimer’s disease,29–32
Parkinson’s disease,33,34 diabetes,35
pancreatitis,36 and cancer.37 One of
the advantages in using an atomic
force microscope for this type ofwork is its ability to perform theseinvestigations in situ.38
Yip and McLaurin used AFM tostudy the mechanisms of amyloid-ß(Aß) fibrillogenesis, which plays arole in Alzheimer’s disease.29 Insitu TappingMode imaging wasperformed on total brain lipid bilayers to study the role of membranecomposition and peptide structure.
Brain lipid bilayers were deposited
onto mica and imaged in phosphate buffer solution (PBS). After

introducing the monomeric Aß peptides into the buffer solution, Aß140 molecules were found to be partially inserted into the bilayer surface (see fig. 5a). After 15 hours,
fibril growth was initiated fromthese sites, resulting in membrane
disruption (see fig. 5b). To study thespecificity of lipid bilayer composition and Aß sequence, the sameexperiment was conducted with
DMPC bilayers and Aß1-28 peptides. From these studies it wasdetermined that the fibril formation
occurs in the presence of acidic
lipids, and that the peptide requires
the hydrophobic C-terminal domain,which is critical for anchoring to thelipid to induce fibrillogenesis.
Without these critical requirements,
membrane disruption is producedby the formation of Aß aggregates
without evidence of fibril formation.
Based on these AFM observations,a better understanding of the mechanisms that result in the Aß fibrillogenesis was formed.Summary
Atomic Force Microscopy (AFM)
has many uses in the field of pharmaceutical research, including theinvestigation of in situ processes,
interaction mechanisms, behavioral
properties, and structure-function
relationships. Although these examples are just a sampling of the workthat has been conducted in druginteraction and disease mechanism
studies, they indicate how importantthe atomic force microscope can bein furthering pharmaceutical sciences. The complementary natureof AFM with other analytical techniques will certainly result in many
more applications as it is further
adopted by the pharmaceuticalcommunity and as new AFM/SPM
techniques are developed.

Figure 5
Images of brain lipid extract bilayers imaged by TappingMode in PBS. After introducing the amyloid-ß peptides (Aß1-40) into the buffer solution, they were found to be partially inserted into thebilayer surface (a). Fibril growth was initiated from these sites, resulting in membrane disruption.
The image in (b) was acquired 15 hours after (a). Images courtesy of C. Yip, University of Toronto.
10µm scan size. [Reference 29]a.)b.)