SCANNING VOL.
32, 61–73 (2010)
& Wiley Periodicals, Inc.
Application of AFM in Microbiology: A Review
SHAOYANG LIU AND YIFEN WANG
Biosystems Engineering Department, Auburn University, Auburn, Alabama
Summary: Atomic force microscopy (AFM) is a
powerful tool for microbiological investigation. This
versatile technique cannot only image cellular sur-
faces at high resolution, but also measure many
forms of fundamental interactions over scales ran-
ging from molecules to cells. In this work, we review
the recent development of AFM applications in the
microbial area. We discuss several approaches for
using AFM scanning images to investigate mor-
phological characteristics of microbes and the use of
force–distance curves to investigate interaction of
microbial samples at the nanometer and cellular
levels. Complementary techniques used in combi-
nation with AFM for study of microbes are also
discussed. SCANNING 32: 61–73, 2010. r 2010
Wiley Periodicals, Inc.
Key words: atomic force microscopy, microbiology,
topography, force–distance curve, tip modification
Introduction
Atomic force microscope (AFM) is a scanning
near-field tool invented in 1986 for nanoscale in-
vestigation (Binnig et al. 1986). Two decades after
the development of AFM, this versatile technique
has been successfully applied in widespread bran-
ches of science and technology such as nanofabri-
cation (Simeone et al. 2009; Tseng et al. 2008),
material science (Bhushan et al. 2008; Johnson 2008;
Withers and Aston 2006), food science (Yang et al.
2007) and microbiology (Cohen and Bitler 2008;
Dufrene 2003, 2008a; Muller et al. 2009).
Address for reprints: Yifen Wang, Biosystems Engineering
Department, 200 Tom E. Corley Building, Auburn University,
Auburn, AL 36849-5417.
E-mail: wangyif@auburn.edu
Received 1 December 2009; Accepted with revision 28 January
2010
DOI 10.1002/sca.20173
Published online 31 March 2010 in Wiley Online Library (wiley
onlinelibrary.com)
The basic idea of AFM is to use a sharp tip
scanning over the surface of a sample while sensing
the interaction between the tip and the sample
(Dufrene 2008a). The tip with a flexible cantilever or
the sample is mounted on a piezoelectric scanner
which can move precisely in three dimensions.
During the test, a laser diode emits a laser beam onto
the back of the cantilever over the tip. As the can-
tilever deflects under load from the laser, the angular
deflection of the reflected laser beam is detected with
a position-sensitive photodiode. Magnitude of the
beam deflection changes in response to the interac-
tion force between the tip and the sample. The AFM
system senses these changes in position and can map
surface topography or monitor the interaction force
between the tip and the sample (Fig. 1).
AFM has some advantages compared with other
forms of microscopes. The optical microscope is a
convenient tool for observation of microbial sam-
ples but its resolution is limited by the wavelength of
the light source to a maximum resolution of ca.
250 nm. Compared with the optical microscope,
AFM has a much higher spatial resolution (up to
sub-nanometer), which provides the ability to map
the distribution of single molecules (Dufrene 2008b;
Engel and Muller 2000). Scanning electron micro-
scopy and transmission electron microscopy can
also provide high resolution, but their complex
sample preparation (e.g. chemical fixing, dehydra-
tion, metal coating and ultrathin section) could
distort the sample substantially. As AFM measures
through direct contact between the tip and the
sample, minimum or even no sample preparation is
required. Furthermore, AFM provides true 3-D
images, whereas only limited ranges in heights can
be ‘‘in-focus’’ at any one time with optical and
electron microscopies. Most importantly for biolo-
gical applications, AFM can investigate samples in
buffer solution, which provides opportunities for
monitoring live cells in real-time.
AFM usually provides two imaging modes,
known as contact mode and dynamic mode, to
visualize topography of microbes. In contact mode,
the AFM tip raster scans over the sample to obtain
high-resolution images (Fig. 2(A)); however, the
62 SCANNING VOL. 32, 2 (2010)
Fig 1. Diagram of AFM work principle.
Fig 2. Diagrams of different AFM operating modes. (A):
Contact mode and (B): dynamic mode for topographic
imaging (Note: The actual oscillation frequency in dynamic
mode is much higher than that in the schematic diagram. It is
much greater than scanning speed so that the tip oscillates
many times at each pixel, and does not hit distant points on
the sample with each oscillation period). (C): Force spectro-
scopy mode for interaction probing.
continuous direct contact between the tip and
sample causes significant lateral force which can
distort soft biological samples. In dynamic mode,
including intermittent contact and noncontact
modes, the cantilever is oscillated near or slightly
above its resonance frequency during the scan
(Martin et al. 1987; Zhong et al. 1993). Conse-
quently, the lateral force between the tip and sample
can be significantly reduced (Fig. 2(B)). Recent ad-
vances in noncontact techniques have led to spatial
resolution up to the atomic level in vacuum and
in liquids (Fukuma et al. 2005; Giessibl 2003;
Sugimoto et al. 2007). Therefore, dynamic mode is
preferred in biological sample analysis.
Some AFM users, including Yang et al. (2008),
further classify the dynamic imaging mode into two
subcategories: intermittent mode and noncontact
mode. In both techniques, the AFM tip is attached
to the end of an oscillating cantilever. For the
intermittent contact technique, the cantilever is
vibrated near its resonance frequency. The ampli-
tude of the oscillation is typically 100–200 nm with
the tip intermittently contacting the sample surface
during the scan. This reduces the force exerted by
the tip on the cell surfaces remarkably in compar-
ison with the contact mode. In the noncontact
mode, the cantilever is vibrated slightly above its
resonance frequency with typical amplitude of sev-
eral nanometers up to less than 10 nm. The tip never
actually contacts the sample surface during the scan,
but van der Waals forces, and other long-range in-
teractions extending above the surface influence
the motion of the tip and provide information on
topography.
Besides topography imaging, AFM can also be
used in force spectroscopy mode to measure inter-
action force and physical properties of biological
samples. In this mode, the cantilever deflection (i.e.
force signal) is recorded as a function of its vertical
displacement (i.e. distance signal) as the tip ap-
proaches toward and retracts from the sample to
obtain a force–distance curve (Fig. 2(C)). Moreover,
spatial resolution can be achieved by generating a
force–volume image through acquiring force–
distance curves at multi-locations. The new frontier
in this area is using specifically functionalized AFM
tips to study protein unfolding/folding mechanisms
and recognize molecular groups on cell surface
(Dufrene 2008a; Muller et al. 2009).
Because of the above-mentioned unique features
AFM has been used in the investigation of micro-
biological samples since the late 1980s, and it has
become a rapidly growing field in the past decade
(Dufrene 2003; Dupres et al. 2009; Hinterdorfer and
Dufrene 2006; Morris et al. 1999). In this work, we
review advances in application of AFM to micro-
biology with the focus mainly on publications
within the last 2–3 years. Different approaches of
using AFM scanning images for morphological
observations and using force–distance curves for
 S. Liu and Y. Wang: Application of AFM in microbiology
interaction investigations are elucidated, and the
combinations of AFM and other complementary
techniques are discussed.
Morphological Observation
One of the most common applications of AFM in
microbiology is to visualize morphology of micro-
organisms. AFM has been widely used to monitor
numerous kinds of microbes and related processes to
provide low-distortion and high-resolution images.
AFM Images from Dried Microbial Samples
Acquisition of AFM images in air is much more
convenient and typically has higher resolution than
that in liquid. Air-dried microbial samples often
provide a suitable hardness for AFM scans without
significant topographic changes; therefore, much
research has been carried out with air/nitrogen-
dried samples.
AFM has been widely applied to evaluate mor-
phological effects of treatments on microbes. To
elucidate the antimicrobial activities of chitosan
with different molecular weight, Eaton et al.
(2008) and Fernandes et al. (2009) investigated
the morphology changes of two Gram-positive
(Staphylococcus aureus and Bacillus cereus) and one
Gram-negative (Escherichia coli) microorganisms
before and after chitosan treatments. Samples were
air-dried on a clean glass surface and scanned with
AFM in air. Chitosans with high molecular weight
surrounded microbe cells and formed a polymer
layer during the treatments. The polymer layer
prevented their uptake of nutrients and eventually
led to their death, which was confirmed by cell wall
collapse observed with AFM, but this effect did not
significantly influence the B. cereus spores as they
can survive for extended periods without nutrients.
On the other hand, chitosans with low molecular
weight (chitooligosaccharides) probably affected
microbes by penetrating the cells, which provoked
more visible damage. Rougher cell surface and lysed
cells were observed with AFM after the treatments,
but the use of chitooligosaccharides by itself on
B. cereus spores was not enough for the destruction
of a large number of cells. The observation also
revealed the response strategies used by the bacteria.
The microbe clusters increased both in amount and
size during the treatments to resist the effects of
chitosans. Liu et al. (2009) evaluated the effects of
ferricyanide on E. coli DH 5 a cell growth and viable
rate. Cell collapse and rough cell surface were
observed with AFM after the bacteria were exposed
in 50 and 100 mM ferricyanide, demonstrating the
63
inhibition effect of ferricyanide on microorganisms.
Dubrovin et al. (2008) developed protocols to in-
vestigate bacteriophage infection of bacterial cells
using AFM and studied different phases of this
process for three types of bacterial hosts: Gram-
negative E. coli 057, Salmonella enteritidis 89, and
Gram-positive B. thuringiensis 393. The whole lytic
cycles of the three hosts, from phage adsorption on
the cells and flagella to complete cell lysis accom-
panied by the release of a large number of newly
formed phages, was observed in AFM images of
infected cells. For example, the AFM images of
E. coli cells during the lytic cycles are shown in
Figure 3. These experiments have demonstrated
AFM is an effective technique for investigation of
phage infection of bacteria, demonstrating its ability
to resolve phage particles in detail, characterize and
compare bacterial surfaces in different phases of
infection, and easily distinguish intact cells from
infected ones.
AFM is also widely used in observation of mi-
croorganism behaviors. Yuan and Pehkonen (2009)
used AFM to study biofilm colonization dynamics
of Pseudomonas NCIMB 2021 and Desulfovibrio
desulfuricans on 304 stainless steel coupon. The re-
sults showed that the biofilm formed on the stainless
steel coupons by the two strains of bacteria in-
creased in coverage, heterogeneity and thickness
with exposure time. The corrosion pits formed by
D. desulfuricans were deeper than that induced by
Pseudomonas NCIMB 2021, which was mainly
attributed to the enhanced corrosion by biogenic
sulfide ions. Zhao et al. (2009) investigated
the morphology of a strain of marine bacteria,
Shewanella sp., on different self-assembled mono-
layers. Interesting fingerprints were observed on
different surfaces. Evidence of morphological
changes and recessed spots surrounding the bacteria
were observed on hexadecanethiol, a hydrophobic
compound monolayer, whereas these phenomena
did not occur on more hydrophilic monolayers.
Hu et al. (2008) carried out a study on anaerobic
degradation of lignin in waste straw by rumen
microorganisms. AFM was employed to image the
treated straw to illustrate the effects of rumen
microbes. The results showed that wax flakelets and
lignin granules covering the straw surface were
removed by the microorganisms and cellulose fibers
located inside the straw were exposed (Fig. 4). This
study provides direct visual evidence to prove the
lignin digestion ability of rumen microbes. After a
careful observation of two strains of E. coli (B and
K12) with AFM, Yang and Wang (2008) proposed
an AFM-based procedure for rapid detection and
quantification of microorganisms in food samples.
This promising method may be useful as an emer-
gency food safety test to help ensure food security.
64 SCANNING VOL. 32, 2 (2010)

Fig 3. AFM images of E. coli cells during its whole lytic cycles caused by phage infection. (A) before mixing with phages, (B–D)
incubated with bacteriophages A157 at 371C (B) for 5 min, (C) for 30 min (insets demonstrate zoomed bacteriophages) and (D) for
60 min. Arrows show the regions of phage release. Insets in panel d present zoomed regions of phage release, indicated by the
arrows. Insets in panel d are height images (height range is 300 nm); others are deflection images. Reprinted with permission from
Dubrovin et al. (2008).

AFM Images of Live Cells
One of the most attractive advantages of AFM
over other nanoscale microscopy is its capability of
monitoring live cells in real time. Typically, a liquid
cell is employed to keep live microbes in buffer so-
lutions. This provides the opportunity for direct
in vitro investigation of microbial processes. Hao
et al. (2009) used AFM to carry out an in situ ob-
servation of Acidiphilium acidophilum on a pyrite
surface in solution during an investigation of phos-
pholipid effects on pyrite oxidation. A syringe pump
was integrated with an AFM-liquid cell to flow so-
lutions across samples during measurements. Dis-
placement of microcolonies was observed after
phospholipid addition, whereas individually bound
bacteria showed little displacement (Fig. 5). The re-
sults revealed that the majority of the bacteria that
are displaced from the pyrite surface were initially
bound in microcolonies. To image dynamic cellular
processes, Kailas et al. (2009) mechanically trapped
coccoid cells of the bacterial species S. aureus in a
lithographically patterned silicon wafer which con-
sists of a square lattice of holes of 450 nm depth
and diameter ranging from 1 to 1.8 mm. Confinement
effects are reduced in this new anchoring approach
compared with trapping the cells in porous mem-
branes or soft gels. The trapped S. aureus cells were
monitored under growth media using AFM and a cell
division was successfully imaged over time. Pepti-
doglycan stretching across the septum was observed
during the division. Many other studies have been
published describing experiments that have employed
live microbial sample imaging with AFM. Most of
them, however, focus more on the analysis of for-
ce–distance curves than surface imaging and will be
discussed in the following section of the review.
Interaction Investigation
Besides morphology imaging, AFM can also be
used to determine the interaction between its tip and
a sample through a force–distance curve acquired in
force spectroscopy mode. This unique ability of
AFM has opened up a new method by which the
physical properties and interactions of biological
samples can be investigated.
 S. Liu and Y. Wang: Application of AFM in microbiology
Fig 4. AFM deflection images of straws treated by rumen
microorganisms for (A) 5 days and (B) 9 days. Arrows in (A)
indicate the exposed cellulose fibers inside the straw.
Reprinted with permission from Hu et al. (2008).
Force Spectroscopy Acquired with Bare AFM Tip
Force–distance curves obtained between a bare
AFM tip and microbe surface can be used to esti-
mate a wide range of physical properties of micro-
organisms. Kumar et al. (2009) studied the changes
of surface-adhesion, indentation depth and Young’s
modulus of a metal-tolerant marine bacterium,
Brevibacterium casei, after its exposure to the Co21
ions during a synthesis of Co3O4 nanocrystals. A set
of force–distance curves was collected with AFM
from the microbial samples during the synthesis
process and these data were used in calculation of
adhesion forces. These forces were found to be 10,
40, 15 and 25 nN after 0, 24, 48 and 72 h of exposure
of the cells to the metal ions during microbial
synthesis, respectively. Indentation depths of
the cells showed an overall increasing trend as the
65
reaction progressed, which was probably due to an
overall decrease in the stiffness of the cell surface
during the synthesis. Young’s modulus was ex-
tremely sensitive to the surrounding environment of
the cells. It tended to decline as the reaction pro-
gressed, showing mostly a decreasing trend in elas-
ticity of the cells. Vadillo-Rodriguez et al. (2008,
2009) used a novel AFM-based technique to in-
vestigate the local viscoelastic properties of in-
dividual gram-negative (Pseudomonas aeruginosa
PAO1 and E. coli) and gram-positive (B. subtilis)
bacterial cells. Time-dependent deflection of the
AFM tip was monitored under constant loading in
fluid conditions. The cells exhibited a viscoelastic
solid-like behavior characterized by an in-
stantaneous elastic deformation followed by a
slower, delayed elastic deformation. The viscoelastic
properties of the bacterial cells were modeled well
using a three-component mechanical model con-
sisting of an elastic spring in series with a parallel
combination of a spring and dashpot. Comparison
of the results obtained for the different strains sug-
gested that the instantaneous elastic response might
be dominated by the properties of the peptidoglycan
layer and the nature of its association with the
membranes, whereas the delayed elastic response
was more likely to arise from the liquid-like char-
acter of the cell membranes. This result will be
helpful in understanding the structure–property
relationships of bacterial cell envelopes, which is
responsible for many important biological func-
tions. Cerf et al. (2009) established a fast, simple and
reproducible procedure to generate a functionalized
pattern for controlled bacteria immobilization based
on a conventional microcontact printing process
and a simple incubation technique. A selective ad-
sorption of bacteria on these local chemical patterns
was achieved, producing highly ordered arrays of
single living bacteria at a success rate close to 100%.
The controlled immobilization method was used to
study the mechanical properties of living and dead
E. coli DH5 a cells in an aqueous environment with
AFM. Young’s modulus of the same cells was
measured using force spectroscopy before and after
heating. The cells with a damaged membrane (after
heating) presented a Young’s modulus twice as high
as that of healthy bacteria. The mechanical prop-
erties provided an additional approach to distin-
guish between a living or a dead cell, something
impossible to do using an AFM topographic image.
Volle et al. (2008) investigated elasticity and adhe-
sion to the AFM tip of cells in five simple biofilms
on glass surfaces formed by three Gram-negative
and two Gram-positive strains. Cellular spring
constants, which represented the cell elasticity,
varied between 0.1670.01 and 0.4170.01 N/m,
where larger spring constants were measured for
66 SCANNING VOL. 32, 2 (2010)

Fig 5. In situ AFM images of a pyrite platelet (A) after a 4 day exposure to a A. ferrooxidans (2  107/ml) containing solution; (B)
10 min, (C) 30 min and (D) 1 h after the introduction of 0.1 mM phospholipid. The solution flow rate through the AFM cell was
0.2 ml/min. The images suggest that microcolonies (enclosed in ovals) were displaced upon phospholipid addition, whereas
individually bound bacteria (enclosed by squares) show little if any displacement. Reprinted with permission from Hao et al. (2009).

Gram-positive cells than for Gram-negative cells.
Adhesive interactions between the retracting silicon
nitride tip and the cells varied between cell types in
terms of the adhesion forces, the adhesion distances
and the number of adhesion events. The Gram-ne-
gative cells’ adhesion to the tip showed the longest
adhesion distance, sometimes more than 1 mm,
whereas the shortest distance adhesion events were
measured between the two Gram-positive cell types
and the tip. These physical properties were related
to the first stages of biofilm formation. The results
showed that all of the biofilm-forming cells had a
high cellular spring constant, indicating they were
quite stiff. It appeared from the extension and re-
traction curves that all of the biofilm-forming cells
were coated by a soft layer of associated extra-
cellular polymeric substances (EPSs). Such EPS
layers are known to facilitate bacterial adhesion to a
surface during biofilm formation, and indeed, they
adhere strongly to the tip as it is retracted. Gabor-
iaud et al. (2008a) measured the interaction forces
between an AFM probe and two types of live She-
wanella bacterial strains with different outer gel-like
layers. The interaction forces were also calculated
through an advanced soft particle electrokinetic
analysis based on electrophoretic mobilities of the
bacteria monitored in the experiments. For both
bacterial strains, the experimental force curves
agreed with the theoretical ones only when the in-
terfacial heterogeneity was considered in the model.
This demonstrated that it is necessary to account for
the spatial distribution of the electrohydrodynamic
parameters in modeling to interpret AFM and
electrokinetic data consistently. It is known that
AFM contact mode imaging does not accurately
locate the apical surface and periphery of the cell
because a lateral component of the applied load on
an AFM tip deforms the cell during the raster scan.
This may cause some difficulty in accurately locat-
ing the cell surface when acquiring a force curve at
an interest point based on a contact mode image. To
solve this problem, Gaboriaud et al. (2008b) em-
ployed a force–volume mode to image live bacterial
cell Shewanella putrefaciens. The mode involved
measurement of a grid of force profiles in which an
individual force–distance curve was acquired at each
pixel and the AFM tip was completely detached
from the cell surface before being moved to the next
pixel. As the tip was only moved vertically when
it contacted the sample (as shown in Fig. 2(C)),
 S. Liu and Y. Wang: Application of AFM in microbiology
no lateral force was applied. Accurately located
force curves were obtained in this study, and me-
chanical properties of the cell (e.g. Young’s mod-
ulus, cell turgor pressure, and elastic and plastic
energies) were extracted from the data of the for-
ce–volume measurements. However, the resolution
of the topographic image, which was reconstructed
from the zero-force height at each probed point on
the cell surface, was very low (10  10 pixels in
4.5  4.5 mm2 area).
Force Spectroscopy Acquired with Functionalized
AFM Tip
Although measurements of the interaction be-
tween a bare AFM tip (typically a silicon nitride tip)
and a sample provides useful information, the ad-
hesion forces may not necessarily reflect the inter-
action involved in real microbial processes,
especially when adhesion forces between cells and a
certain chemical group, molecule or surface are of
interest. To monitor interaction forces of interest,
modified AFM tips were introduced. AFM tips can
be functionalized using specific functional groups,
molecules or even cells. Based on the materials used
to coat the tip, these corresponding techniques are
called chemical force microscopy (CFM), single-
molecule force spectroscopy (SMFS) and single-cell
force spectroscopy (SCFS). This development has
greatly extended applications of AFM in micro-
biology.
In CFM, AFM tips are modified with specific
functional groups to monitor microscale interac-
tions (Frisbie et al. 1994, Noy 2006). Alsteens et al.
(2007) used CFM with hydrophobic, methyl-termi-
nated tips to measure local hydrophobic forces on
organic surfaces with different hydrophobicities.
The results were well correlated with the macroscale
wettability of the surfaces determined by water
contact angle, demonstrating the ability of CFM for
sensing microscale hydrophobic interactions. The
hydrophobic tip was then used to probe the surface
of mycobacteria. Strong hydrophobicity was found
on the surface, which may have been due to the
presence of the hydrophobic mycolic acid in the
outermost layer. The discovery supports the notion
that these hydrophobic compounds represent an
important permeation barrier to drugs. Dague et al.
(2007, 2008) employed the same hydrophobic tips to
map the hydrophobicity of a live human opportu-
nistic pathogen, Aspergillus fumigatus, at the na-
noscale. The conidial surface in an aqueous solution
displayed densely packed rodlets and was uniformly
hydrophobic. Moreover, continuous observation on
a single spore during its germination was achieved,
and a substantial reduction of adhesion contrast
67
was noted, reflecting a dramatic decrease of hydro-
phobicity (Fig. 6). In another article from the same
research group, Dufrene (2008c) presented proto-
cols for analyzing spores of the pathogen A. fumi-
gatus using real-time AFM imaging and CFM. The
use of porous polymer membranes for immobilizing
single live cells and the modification of gold-coated
tips with alkanethiols for CFM measurements were
discussed, as well as the recording conditions and
data interpretation.
Dorobantu et al. (2008, 2009) investigated two
live bacterial species, Acinetobacter Venetianus
RAG-1 and Rhodococcus erythropolis 20S-E1-c.
A hydrophobic AFM tip functionalized to expose
methyl groups was used to measure adhesion forces
on the cell surfaces. A. Venetianus RAG-1 showed
an irregular pattern with multiple adhesion peaks
suggesting the presence of biopolymers with differ-
ent lengths on its surface. R. erythropolis 20S-E1-c
exhibited long-range attraction forces and single
rupture events suggesting a more hydrophobic and
smoother surface. Heterogeneity at a length scale of
50–100 nm was observed on the surfaces of both
strains. As the force curves were not successfully
described by the classical Derjaguin–Landau–
Verwey–Overbeek (DLVO) theory, two types of
extended DLVO models were proposed. The first
modification considers an additional acid–base
component that accounts for attractive hydrophobic
interactions and repulsive hydration effects, and the
second model considers an additional exponentially
decaying steric interaction between polymeric bru-
shes in addition to the acid–base interactions. The
predictions of those extended DLVO models agreed
well with the AFM experimental data, indicating
that bacterial adhesion is significantly influenced by
the presence of extracellular structures. Herzberg
et al. (2009) studied the interactions between a car-
boxylate-modified latex (CML) particle functiona-
lized AFM tip and an EPS-fouled reverse osmosis
(RO) membrane surface. A significant increase of
the adhesion force was observed with the presence
of calcium ions, indicating a bridging effect between
the EPS and the CML probe due to the complexa-
tion or binding of calcium ions to carboxylic groups.
This effect may play a major role in biofouling of
RO membranes.
In SMFS, AFM tips are modified using mole-
cules with specific biochemical activities to char-
acterize molecular interactions and distributions
in biological systems (Muller and Dufrene 2008;
Neuman and Nagy 2008). Francius et al. (2008)
investigated the clinically important probiotic
bacterium Lactobacillus rhamnosus GG (LGG)
wild-type and the CMPG5413 mutant which
was impaired in adherence, biofilm formation
and polysaccharide production. Cell surface
68 SCANNING VOL. 32, 2 (2010)

Fig 6. High-resolution deflection images (left) and adhesion force maps (right, scale bars: 100 nm; z range 5 5 nN) recorded on a
single A. fumigatus spore during germination. The conidial surface had a crystalline rodlet look and was uniformly hydrophobic
before the germination. After 2 h, both rodlet and amorphous regions were found to coexist (separated by dash line), and
heterogeneous contrast was observed in the form of hydrophobic patches surrounded by a hydrophilic sea. Within 3 h, the
crystalline rodlet layer changed into a layer of amorphous material. The substantial reduction of adhesion contrast reflected a
dramatic decrease of hydrophobicity. Reprinted with permission from Dague et al. (2008).

polysaccharides were probed at the molecular level
to elucidate the differences. For this purpose, AFM
tips were functionalized to expose the lectin con-
canavalin A (Con A) and the lectin from P. aeru-
ginosa (PA-I) via a 6 nm-long polyethylene glycol
chain. Two kinds of polysaccharide chains were
identified on the cell surfaces of both strains: poly-
saccharides rich in mannose (recognized by Con
A-terminated tips) having moderate extensions,
and polysaccharides rich in galactose (recognized
by PA-I-terminated tips) having much longer
extensions. The polysaccharide chains on LGG
wild-type had much larger adhesion forces, longer
rupture distances, and were more densely dis-
tributed compared to those of the mutant. This
study demonstrated the crucial role played by cell
wall polysaccharides in determining nanoscale sur-
face properties. Alsteens et al. (2008) studied two
brewing yeast strains, Saccharomyces carlsbergensis
and S. cerevisiae with in situ AFM in three different
modes. Although both strains are smooth and
homogeneous, an evident difference in cell wall
 S. Liu and Y. Wang: Application of AFM in microbiology 69

elasticity was revealed in nanomechanical measure-
ments (Fig. 7(A–D)). In SMFS analysis with Con
A-modified tips, major differences in polysaccharide
properties of the two strains were discovered. Poly-
saccharides on S. cerevisiae surface were more ex-
tended, suggesting that not only oligosaccharides but
also polypeptide chains of the mannoproteins were
stretched (Fig. 7(E, F)). These findings at the nanoscale
and molecular level are useful to explain the different
aggregation properties of the two organisms.
Another remarkable application of SMFS is to
study unfolding forces and mechanics of proteins.
The pioneering work in measuring mechanical
properties and unfolding force of individual titin
molecules was achieved with SMFS in the late 1990s
(Rief et al. 1997). These molecular mechanisms of

Fig 7. AFM images recorded on bud scar region of S. carlsbergensis (left) and S. cerevisiae (right). (A), (B): deflection images;
(C), (D): corresponding elasticity maps (z-range 5 3 MPa); (E), (F): three-dimensional reconstructed maps of polymer properties
obtained by combining adhesion force values (expressed as false colors) and rupture distance (expressed as z level). Reprinted with
permission from Alsteens et al. (2008).
70 SCANNING VOL. 32, 2 (2010)
cell adhesion proteins are important for the identi-
fication of potential drug targets. Alsteens et al.
(2009) explored the adhesive and mechanical prop-
erties of the widely expressed Als5p cell adhesion
protein from the opportunistic pathogen Candida
albicans. The forces required to unfold individual
tandem repeats of the protein were determined to be
150–250 pN. The unfolding probability increased
with the number of tandem repeats and correlated
with the level of cell adherence. The authors sug-
gested that this modular elongation mechanism
imparted both strength and toughness to the pro-
tein, making it ideally suited to function as an ad-
hesion molecule.
In SCFS, AFM tips are replaced or modified with
one or more living cells that are used to measure
interactions toward other cells or substrates
(Helenius et al. 2008). The force–distance curve re-
corded when pulling the cell back from its objective
can detect different unbinding events and discrete
force steps can be assigned to the rupture of
single cell adhesion molecules. Boks et al. (2008)
investigated adhesion forces of four strains of
S. epidermidis to hydrophobic and hydrophilic sur-
faces. Significant differences of initial adhesion for-
ces were observed between the two surfaces.
Over time, bond strengthening on hydrophobic
dimethyldichlorosilane-coated glass was fast (less
than 10 s), limited to a minor increase in adhesion
force, and likely governed by hydrophobic interac-
tion. However, bond strengthening on hydrophilic
glass occurred within 5–35 s to maximum adhesion
forces of 1.970.7 nN and was concurrent with the
development of multiple adhesion peaks upon
retract. This was attributed to progressive formation
of hydrogen bonds made possible by ongoing
rearrangements of outer cell surface structures. As a
consequence, adhesion forces strengthened con-
siderably more on hydrophilic glass than on
hydrophobic glass. Poisson analysis of the multiple
adhesion peaks allowed separation of contributions
of hydrogen bonding from other nonspecific inter-
action forces and revealed a force contribution of
0.8 nN for hydrogen bonding and 10.3 nN for
other nonspecific interaction forces. The time-de-
pendent bacterial adhesion forces were comparable
for all four staphylococcal strains. Kang and
Elimelech (2009) developed a novel procedure for
preparing live bacterial cell probes for AFM using a
bioinspired polydopamine wet adhesive. Traditional
adhesion methods, which use various chemical ap-
proaches, likely denature bacterial surfaces by cross-
linking proteins during glutaraldehyde treatment,
rearrange bacterial cell surface structure by the ad-
sorption of cells onto positively charged surfaces,
and inactivate cells glued to the end of the canti-
lever. The new method could keep the attached cells
alive during the force spectrum measurements. Re-
sults showed that bacterial interactions with quartz
surfaces measured with probes prepared through the
new method were greatly different from those ob-
tained with the glutaraldehyde-treated cell probes.
Great influence by the bacterium exocellular poly-
mers and solution chemistry on the interactions was
observed.
Combining AFM with Other Analytical
Techniques
Although AFM is a powerful technique with
many advantages, this stand-alone tool has its own
limitations. Combinations of AFM with other
complementary techniques provide great opportu-
nities for obtaining more comprehensive informa-
tion from interesting samples, which further
expands AFM applications in microbiology (Flores
and Toca-Herrera 2009; Oreopoulos and Yip 2008).
Combining AFM with Optical Microscopy
As a sensing technique, AFM uses its tip to ‘‘feel’’
samples (Morris et al. 1999). Combination of AFM
with transmitted light optical microscopes, e.g.
fluorescence lifetime imaging microscopy (FLIM)
and total internal reflection fluorescence microscopy
(TIRFM), can enable researchers to ‘‘look’’ at the
samples simultaneously.
In biological applications, organelles and mole-
cular complexes can be selectively labeled with
fluorescent dyes to make them visible. The combi-
nation of AFM topography and fluorescence images
provides a unique tool for correlating organelles and
chemical components with cell morphology. Micic
et al. (2004) carried out an investigation on living
Gram-negative S. oneidensis MR-1 cells with high-
resolution AFM-tip-enhanced FLIM. A gene fusion
of yellow fluorescent protein (YFP) to the methyl
accepting chemotaxis protein (MCP) was con-
structed before the test. The AFM height image,
fluorescence intensity image and fluorescence life-
time image of single cells in the same sample region
are shown in Figure 8(A–C), respectively. The AFM
image showed higher surface topography at both
ends of the cells. Fluorescence images also showed
higher intensities at both ends of the cells, which
suggests polar localization of the MCP-YFP fusion
protein. Therefore, a correlation between the sur-
face protuberances in MR-1 cells and polar locali-
zation of the MCP-YFP fusion protein could be
concluded based on the combination of the AFM
and fluorescence images.
 S. Liu and Y. Wang: Application of AFM in microbiology
Fig 8. AFM-confocal FLIM image of S. oneidensis bacterial cells on poly-L-lysine surface: (A) topographic AFM image, (B)
confocal fluorescence intensity and (C) confocal fluorescence lifetime image. The differences of the fluorescence intensity and
lifetime are presented by the brightness and color (inset), respectively. The ploy-L-lysine was used as an immobilizer of the cells.
Reprinted with permission from Micic et al. (2004).
TIRFM uses evanescent waves to selectively
illuminate and excite fluorophores in a restricted
region of the specimen immediately below the total
reflection interface (typically a few tens of nan-
ometers). As a consequence, a high-resolution
fluorescence image of a sample surface can be ob-
tained. Shaw et al. (2006, 2008) investigated how a
cationic antimicrobial peptide, indolicidin, inter-
acted with model membranes, mixed zwitterionic
planar lipid bilayers, with AFM-confocal-TIRFM.
The results indicated that the peptide rearranged the
model membranes in a concentration- and lipid-
dependent manner. At low peptide concentration, it
appeared to reduce the interfacial line tension at the
domain boundary between the liquid-ordered and
liquid-disordered domains. Peptide-induced mem-
brane remodeling only occurred under high peptide
concentration. These works demonstrated that cor-
related AFM-confocal-TIRFM imaging is an at-
tractive approach for tracking preferential
localization of fluorescent lipid probes and well
suited for tracking and characterizing protein–
membrane interactions.
Combining AFM with Tip-Enhanced Raman Spectro-
scopy
AFM alone is not sufficient to provide informa-
tion on the chemical identity of features on a sur-
face. Tip-enhanced Raman spectroscopy (TERS) is
a label-free nanoscale chemical characterization
technique. It uses a sharp metal tip (e.g. Ag or Au).
When the tip is sufficiently close (ca. 1 nm) to the
sample, the near field can optically excite nearby
molecules on the sample surface, and thus, local
Raman spectra can be obtained. Combining AFM
with TERS should be a promising way to improve
chemical recognition ability for AFM. Schmid
et al. (2008) employed a well-defined model system
71
consisting of calcium alginate fibers to evaluate the
feasibility of the combination of AFM and TERS in
biological systems. Alginates are stabilizing com-
pounds in the extracellular polymeric substances of
certain biofilms. The mainly guluronic acid-rich
parts of alginate can interact with Ca21 ions and
cross-link different alginate strands resulting in a
three dimensional network-like structure, which is a
good model system for investigation of microbial
aggregates. The investigation of calcium alginate
fibers showed that Raman frequencies in TERS
spectra of biopolymers do not necessarily resemble
band positions in the normal Raman spectrum of
the bulk material. Additionally, analyte decom-
position due to laser heating and carbon con-
tamination were observed. Fortunately, strategies
for spectra correction, choice of appropriate
reference samples and data interpretation were
presented by the authors. With this approach,
characteristic frequency ranges and specific marker
bands can be found for biological macromolecules,
which can be employed for their identification in
complex environments.
Conclusions
AFM is a versatile tool that images cellular sur-
faces at high resolution and also measures funda-
mental interactions giving cells their characteristic
structure–function relationship (Muller et al. 2009).
Although a topography measurement of air-dried
samples is still the most common application of
AFM in microbiology, morphology probing on
living cells will be more frequently employed in the
future given improvements in the instrument and
operators’ skill. Besides imaging, force–distance
curves generated using AFM show great potential
for elucidating microbial interactions and physical
properties. Through modification of the AFM tip, it
is possible to investigate many forms of interactions
72 SCANNING VOL. 32, 2 (2010)
over scales ranging from molecules to cells. The
applications of CFM, SMFS and SCFS have been
dramatically expanded in the last few years and will
remain a focus of investigation in the near future.
Combinations of AFM with other complementary
techniques are being considered by more and more
researchers and developments in this area will open
new ways to look at biological samples. In sum-
mary, AFM-based measurements will continue to
play an important role in future microbiological
research, and improve our understanding of the
structures and properties of microbial cell surfaces
and the mechanisms involved in microbial behavior
at the molecular level.
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