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 apowerful tool for microbiological investigation. Thisversatile technique cannot only image cellular sur-faces at high resolution, but also measure manyforms of fundamental interactions over scales ran-ging from molecules to cells. In this work, we reviewthe recent development of AFM applications in themicrobial area. We discuss several approaches forusing AFM scanning images to investigate mor-phological characteristics of microbes and the use offorce–distance curves to investigate interaction ofmicrobial samples at the nanometer and cellularlevels. Complementary techniques used in combi-nation with AFM for study of microbes are alsodiscussed. SCANNING 32: 61–73, 2010. r 2010Wiley Periodicals, Inc.

Key words: atomic force microscopy, microbiology,topography, force–distance curve, tip modification

Introduction

Atomic force microscope (AFM) is a scanningnear-field tool invented in 1986 for nanoscale in-vestigation (Binnig et al. 1986). Two decades afterthe development of AFM, this versatile techniquehas 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).

The basic idea of AFM is to use a sharp tipscanning over the surface of a sample while sensingthe interaction between the tip and the sample(Dufrene 2008a). The tip with a flexible cantilever orthe sample is mounted on a piezoelectric scannerwhich can move precisely in three dimensions.During the test, a laser diode emits a laser beam ontothe back of the cantilever over the tip. As the can-tilever deflects under load from the laser, the angulardeflection of the reflected laser beam is detected witha position-sensitive photodiode. Magnitude of thebeam deflection changes in response to the interac-tion force between the tip and the sample. The AFMsystem senses these changes in position and can mapsurface topography or monitor the interaction forcebetween the tip and the sample (Fig. 1).

AFM has some advantages compared with otherforms of microscopes. The optical microscope is aconvenient tool for observation of microbial sam-ples but its resolution is limited by the wavelength ofthe light source to a maximum resolution of ca.250 nm. Compared with the optical microscope,AFM has a much higher spatial resolution (up tosub-nanometer), which provides the ability to mapthe distribution of single molecules (Dufrene 2008b;Engel and Muller 2000). Scanning electron micro-scopy and transmission electron microscopy canalso provide high resolution, but their complexsample preparation (e.g. chemical fixing, dehydra-tion, metal coating and ultrathin section) coulddistort the sample substantially. As AFM measuresthrough direct contact between the tip and thesample, minimum or even no sample preparation isrequired. Furthermore, AFM provides true 3-Dimages, whereas only limited ranges in heights canbe ‘‘in-focus’’ at any one time with optical andelectron microscopies. Most importantly for biolo-gical applications, AFM can investigate samples inbuffer solution, which provides opportunities formonitoring live cells in real-time.

AFM usually provides two imaging modes,known as contact mode and dynamic mode, tovisualize topography of microbes. In contact mode,the AFM tip raster scans over the sample to obtainhigh-resolution images (Fig. 2(A)); however, the

DOI 10.1002/sca.20173

Published online 31 March 2010 in Wiley Online Library (wiley

onlinelibrary.com)

Received 1 December 2009; Accepted with revision 28 January

2010

Address for reprints: Yifen Wang, Biosystems Engineering

Department, 200 Tom E. Corley Building, Auburn University,

Auburn, AL 36849-5417.

E-mail: wangyif@auburn.edu

SCANNING VOL. 32, 61–73 (2010)& Wiley Periodicals, Inc.

continuous direct contact between the tip andsample causes significant lateral force which candistort soft biological samples. In dynamic mode,

including intermittent contact and noncontactmodes, the cantilever is oscillated near or slightlyabove its resonance frequency during the scan(Martin et al. 1987; Zhong et al. 1993). Conse-quently, the lateral force between the tip and samplecan be significantly reduced (Fig. 2(B)). Recent ad-vances in noncontact techniques have led to spatialresolution up to the atomic level in vacuum andin liquids (Fukuma et al. 2005; Giessibl 2003;Sugimoto et al. 2007). Therefore, dynamic mode ispreferred in biological sample analysis.

Some AFM users, including Yang et al. (2008),further classify the dynamic imaging mode into twosubcategories: intermittent mode and noncontactmode. In both techniques, the AFM tip is attachedto the end of an oscillating cantilever. For theintermittent contact technique, the cantilever isvibrated near its resonance frequency. The ampli-tude of the oscillation is typically 100–200 nm withthe tip intermittently contacting the sample surfaceduring the scan. This reduces the force exerted bythe tip on the cell surfaces remarkably in compar-ison with the contact mode. In the noncontactmode, the cantilever is vibrated slightly above itsresonance frequency with typical amplitude of sev-eral nanometers up to less than 10 nm. The tip neveractually contacts the sample surface during the scan,but van der Waals forces, and other long-range in-teractions extending above the surface influencethe motion of the tip and provide information ontopography.

Besides topography imaging, AFM can also beused in force spectroscopy mode to measure inter-action force and physical properties of biologicalsamples. In this mode, the cantilever deflection (i.e.force signal) is recorded as a function of its verticaldisplacement (i.e. distance signal) as the tip ap-proaches toward and retracts from the sample toobtain a force–distance curve (Fig. 2(C)). Moreover,spatial resolution can be achieved by generating aforce–volume image through acquiring force–distance curves at multi-locations. The new frontierin this area is using specifically functionalized AFMtips to study protein unfolding/folding mechanismsand recognize molecular groups on cell surface(Dufrene 2008a; Muller et al. 2009).

Because of the above-mentioned unique featuresAFM has been used in the investigation of micro-biological samples since the late 1980s, and it hasbecome a rapidly growing field in the past decade(Dufrene 2003; Dupres et al. 2009; Hinterdorfer andDufrene 2006; Morris et al. 1999). In this work, wereview advances in application of AFM to micro-biology with the focus mainly on publicationswithin the last 2–3 years. Different approaches ofusing AFM scanning images for morphologicalobservations and using force–distance curves for

Fig 2. Diagrams of different AFM operating modes. (A):Contact mode and (B): dynamic mode for topographicimaging (Note: The actual oscillation frequency in dynamicmode is much higher than that in the schematic diagram. It ismuch greater than scanning speed so that the tip oscillatesmany times at each pixel, and does not hit distant points onthe sample with each oscillation period). (C): Force spectro-scopy mode for interaction probing.

Fig 1. Diagram of AFM work principle.

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interaction investigations are elucidated, and thecombinations of AFM and other complementarytechniques are discussed.

Morphological Observation

One of the most common applications of AFM inmicrobiology is to visualize morphology of micro-organisms. AFM has been widely used to monitornumerous kinds of microbes and related processes toprovide low-distortion and high-resolution images.

AFM Images from Dried Microbial Samples

Acquisition of AFM images in air is much moreconvenient and typically has higher resolution thanthat in liquid. Air-dried microbial samples oftenprovide a suitable hardness for AFM scans withoutsignificant topographic changes; therefore, muchresearch has been carried out with air/nitrogen-dried samples.

AFM has been widely applied to evaluate mor-phological effects of treatments on microbes. Toelucidate the antimicrobial activities of chitosanwith different molecular weight, Eaton et al.(2008) and Fernandes et al. (2009) investigatedthe morphology changes of two Gram-positive(Staphylococcus aureus and Bacillus cereus) and oneGram-negative (Escherichia coli) microorganismsbefore and after chitosan treatments. Samples wereair-dried on a clean glass surface and scanned withAFM in air. Chitosans with high molecular weightsurrounded microbe cells and formed a polymerlayer during the treatments. The polymer layerprevented their uptake of nutrients and eventuallyled to their death, which was confirmed by cell wallcollapse observed with AFM, but this effect did notsignificantly influence the B. cereus spores as theycan survive for extended periods without nutrients.On the other hand, chitosans with low molecularweight (chitooligosaccharides) probably affectedmicrobes by penetrating the cells, which provokedmore visible damage. Rougher cell surface and lysedcells were observed with AFM after the treatments,but the use of chitooligosaccharides by itself onB. cereus spores was not enough for the destructionof a large number of cells. The observation alsorevealed the response strategies used by the bacteria.The microbe clusters increased both in amount andsize during the treatments to resist the effects ofchitosans. Liu et al. (2009) evaluated the effects offerricyanide on E. coliDH 5 a cell growth and viablerate. Cell collapse and rough cell surface wereobserved with AFM after the bacteria were exposedin 50 and 100 mM ferricyanide, demonstrating the

inhibition effect of ferricyanide on microorganisms.Dubrovin et al. (2008) developed protocols to in-vestigate bacteriophage infection of bacterial cellsusing AFM and studied different phases of thisprocess for three types of bacterial hosts: Gram-negative E. coli 057, Salmonella enteritidis 89, andGram-positive B. thuringiensis 393. The whole lyticcycles of the three hosts, from phage adsorption onthe cells and flagella to complete cell lysis accom-panied by the release of a large number of newlyformed phages, was observed in AFM images ofinfected cells. For example, the AFM images ofE. coli cells during the lytic cycles are shown inFigure 3. These experiments have demonstratedAFM is an effective technique for investigation ofphage infection of bacteria, demonstrating its abilityto resolve phage particles in detail, characterize andcompare bacterial surfaces in different phases ofinfection, and easily distinguish intact cells frominfected ones.

AFM is also widely used in observation of mi-croorganism behaviors. Yuan and Pehkonen (2009)used AFM to study biofilm colonization dynamicsof Pseudomonas NCIMB 2021 and Desulfovibriodesulfuricans on 304 stainless steel coupon. The re-sults showed that the biofilm formed on the stainlesssteel coupons by the two strains of bacteria in-creased in coverage, heterogeneity and thicknesswith exposure time. The corrosion pits formed byD. desulfuricans were deeper than that induced byPseudomonas NCIMB 2021, which was mainlyattributed to the enhanced corrosion by biogenicsulfide ions. Zhao et al. (2009) investigatedthe morphology of a strain of marine bacteria,Shewanella sp., on different self-assembled mono-layers. Interesting fingerprints were observed ondifferent surfaces. Evidence of morphologicalchanges and recessed spots surrounding the bacteriawere observed on hexadecanethiol, a hydrophobiccompound monolayer, whereas these phenomenadid not occur on more hydrophilic monolayers.Hu et al. (2008) carried out a study on anaerobicdegradation of lignin in waste straw by rumenmicroorganisms. AFM was employed to image thetreated straw to illustrate the effects of rumenmicrobes. The results showed that wax flakelets andlignin granules covering the straw surface wereremoved by the microorganisms and cellulose fiberslocated inside the straw were exposed (Fig. 4). Thisstudy provides direct visual evidence to prove thelignin digestion ability of rumen microbes. After acareful observation of two strains of E. coli (B andK12) with AFM, Yang and Wang (2008) proposedan AFM-based procedure for rapid detection andquantification of microorganisms in food samples.This promising method may be useful as an emer-gency food safety test to help ensure food security.

S. Liu and Y. Wang: Application of AFM in microbiology 63

AFM Images of Live Cells

One of the most attractive advantages of AFMover other nanoscale microscopy is its capability ofmonitoring live cells in real time. Typically, a liquidcell is employed to keep live microbes in buffer so-lutions. This provides the opportunity for directin vitro investigation of microbial processes. Haoet al. (2009) used AFM to carry out an in situ ob-servation of Acidiphilium acidophilum on a pyritesurface in solution during an investigation of phos-pholipid effects on pyrite oxidation. A syringe pumpwas integrated with an AFM-liquid cell to flow so-lutions across samples during measurements. Dis-placement of microcolonies was observed afterphospholipid addition, whereas individually boundbacteria showed little displacement (Fig. 5). The re-sults revealed that the majority of the bacteria thatare displaced from the pyrite surface were initiallybound in microcolonies. To image dynamic cellularprocesses, Kailas et al. (2009) mechanically trappedcoccoid cells of the bacterial species S. aureus in alithographically 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. Confinementeffects are reduced in this new anchoring approachcompared with trapping the cells in porous mem-branes or soft gels. The trapped S. aureus cells weremonitored under growth media using AFM and a celldivision was successfully imaged over time. Pepti-doglycan stretching across the septum was observedduring the division. Many other studies have beenpublished describing experiments that have employedlive microbial sample imaging with AFM. Most ofthem, however, focus more on the analysis of for-ce–distance curves than surface imaging and will bediscussed in the following section of the review.

Interaction Investigation

Besides morphology imaging, AFM can also beused to determine the interaction between its tip anda sample through a force–distance curve acquired inforce spectroscopy mode. This unique ability ofAFM has opened up a new method by which thephysical properties and interactions of biologicalsamples can be investigated.

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) for60 min. Arrows show the regions of phage release. Insets in panel d present zoomed regions of phage release, indicated by thearrows. Insets in panel d are height images (height range is 300 nm); others are deflection images. Reprinted with permission fromDubrovin et al. (2008).

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Force Spectroscopy Acquired with Bare AFM Tip

Force–distance curves obtained between a bareAFM 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 changesof surface-adhesion, indentation depth and Young’smodulus of a metal-tolerant marine bacterium,Brevibacterium casei, after its exposure to the Co21

ions during a synthesis of Co3O4 nanocrystals. A setof force–distance curves was collected with AFMfrom the microbial samples during the synthesisprocess and these data were used in calculation ofadhesion forces. These forces were found to be 10,40, 15 and 25 nN after 0, 24, 48 and 72 h of exposureof the cells to the metal ions during microbialsynthesis, respectively. Indentation depths ofthe cells showed an overall increasing trend as the

reaction progressed, which was probably due to anoverall decrease in the stiffness of the cell surfaceduring the synthesis. Young’s modulus was ex-tremely sensitive to the surrounding environment ofthe 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 aeruginosaPAO1 and E. coli) and gram-positive (B. subtilis)bacterial cells. Time-dependent deflection of theAFM tip was monitored under constant loading influid conditions. The cells exhibited a viscoelasticsolid-like behavior characterized by an in-stantaneous elastic deformation followed by aslower, delayed elastic deformation. The viscoelasticproperties of the bacterial cells were modeled wellusing a three-component mechanical model con-sisting of an elastic spring in series with a parallelcombination of a spring and dashpot. Comparisonof the results obtained for the different strains sug-gested that the instantaneous elastic response mightbe dominated by the properties of the peptidoglycanlayer and the nature of its association with themembranes, whereas the delayed elastic responsewas more likely to arise from the liquid-like char-acter of the cell membranes. This result will behelpful in understanding the structure–propertyrelationships of bacterial cell envelopes, which isresponsible for many important biological func-tions. Cerf et al. (2009) established a fast, simple andreproducible procedure to generate a functionalizedpattern for controlled bacteria immobilization basedon a conventional microcontact printing processand a simple incubation technique. A selective ad-sorption of bacteria on these local chemical patternswas achieved, producing highly ordered arrays ofsingle living bacteria at a success rate close to 100%.The controlled immobilization method was used tostudy the mechanical properties of living and deadE. coli DH5 a cells in an aqueous environment withAFM. Young’s modulus of the same cells wasmeasured using force spectroscopy before and afterheating. The cells with a damaged membrane (afterheating) presented a Young’s modulus twice as highas that of healthy bacteria. The mechanical prop-erties provided an additional approach to distin-guish between a living or a dead cell, somethingimpossible 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 biofilmson glass surfaces formed by three Gram-negativeand two Gram-positive strains. Cellular springconstants, which represented the cell elasticity,varied between 0.1670.01 and 0.4170.01 N/m,where larger spring constants were measured for

Fig 4. AFM deflection images of straws treated by rumenmicroorganisms 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).

S. Liu and Y. Wang: Application of AFM in microbiology 65

Gram-positive cells than for Gram-negative cells.Adhesive interactions between the retracting siliconnitride tip and the cells varied between cell types interms of the adhesion forces, the adhesion distancesand the number of adhesion events. The Gram-ne-gative cells’ adhesion to the tip showed the longestadhesion distance, sometimes more than 1 mm,whereas the shortest distance adhesion events weremeasured between the two Gram-positive cell typesand the tip. These physical properties were relatedto the first stages of biofilm formation. The resultsshowed that all of the biofilm-forming cells had ahigh cellular spring constant, indicating they werequite stiff. It appeared from the extension and re-traction curves that all of the biofilm-forming cellswere coated by a soft layer of associated extra-cellular polymeric substances (EPSs). Such EPSlayers are known to facilitate bacterial adhesion to asurface during biofilm formation, and indeed, theyadhere strongly to the tip as it is retracted. Gabor-iaud et al. (2008a) measured the interaction forcesbetween an AFM probe and two types of live She-wanella bacterial strains with different outer gel-likelayers. The interaction forces were also calculatedthrough an advanced soft particle electrokinetic

analysis based on electrophoretic mobilities of thebacteria monitored in the experiments. For bothbacterial strains, the experimental force curvesagreed with the theoretical ones only when the in-terfacial heterogeneity was considered in the model.This demonstrated that it is necessary to account forthe spatial distribution of the electrohydrodynamicparameters in modeling to interpret AFM andelectrokinetic data consistently. It is known thatAFM contact mode imaging does not accuratelylocate the apical surface and periphery of the cellbecause a lateral component of the applied load onan 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 atan interest point based on a contact mode image. Tosolve this problem, Gaboriaud et al. (2008b) em-ployed a force–volume mode to image live bacterialcell Shewanella putrefaciens. The mode involvedmeasurement of a grid of force profiles in which anindividual force–distance curve was acquired at eachpixel and the AFM tip was completely detachedfrom the cell surface before being moved to the nextpixel. As the tip was only moved vertically whenit contacted the sample (as shown in Fig. 2(C)),

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 was0.2 ml/min. The images suggest that microcolonies (enclosed in ovals) were displaced upon phospholipid addition, whereasindividually bound bacteria (enclosed by squares) show little if any displacement. Reprinted with permission from Hao et al. (2009).

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no lateral force was applied. Accurately locatedforce 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 plasticenergies) were extracted from the data of the for-ce–volume measurements. However, the resolutionof the topographic image, which was reconstructedfrom the zero-force height at each probed point onthe cell surface, was very low (10� 10 pixels in4.5� 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 acertain chemical group, molecule or surface are ofinterest. To monitor interaction forces of interest,modified AFM tips were introduced. AFM tips canbe functionalized using specific functional groups,molecules or even cells. Based on the materials usedto coat the tip, these corresponding techniques arecalled chemical force microscopy (CFM), single-molecule force spectroscopy (SMFS) and single-cellforce spectroscopy (SCFS). This development hasgreatly extended applications of AFM in micro-biology.

In CFM, AFM tips are modified with specificfunctional 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 onorganic surfaces with different hydrophobicities.The results were well correlated with the macroscalewettability of the surfaces determined by watercontact angle, demonstrating the ability of CFM forsensing microscale hydrophobic interactions. Thehydrophobic tip was then used to probe the surfaceof mycobacteria. Strong hydrophobicity was foundon the surface, which may have been due to thepresence of the hydrophobic mycolic acid in theoutermost layer. The discovery supports the notionthat these hydrophobic compounds represent animportant permeation barrier to drugs. Dague et al.(2007, 2008) employed the same hydrophobic tips tomap the hydrophobicity of a live human opportu-nistic pathogen, Aspergillus fumigatus, at the na-noscale. The conidial surface in an aqueous solutiondisplayed densely packed rodlets and was uniformlyhydrophobic. Moreover, continuous observation ona single spore during its germination was achieved,and a substantial reduction of adhesion contrast

was noted, reflecting a dramatic decrease of hydro-phobicity (Fig. 6). In another article from the sameresearch group, Dufrene (2008c) presented proto-cols for analyzing spores of the pathogen A. fumi-gatus using real-time AFM imaging and CFM. Theuse of porous polymer membranes for immobilizingsingle live cells and the modification of gold-coatedtips with alkanethiols for CFM measurements werediscussed, as well as the recording conditions anddata interpretation.

Dorobantu et al. (2008, 2009) investigated twolive bacterial species, Acinetobacter VenetianusRAG-1 and Rhodococcus erythropolis 20S-E1-c.A hydrophobic AFM tip functionalized to exposemethyl groups was used to measure adhesion forceson the cell surfaces. A. Venetianus RAG-1 showedan irregular pattern with multiple adhesion peakssuggesting the presence of biopolymers with differ-ent lengths on its surface. R. erythropolis 20S-E1-cexhibited long-range attraction forces and singlerupture events suggesting a more hydrophobic andsmoother surface. Heterogeneity at a length scale of50–100 nm was observed on the surfaces of bothstrains. As the force curves were not successfullydescribed by the classical Derjaguin–Landau–Verwey–Overbeek (DLVO) theory, two types ofextended DLVO models were proposed. The firstmodification considers an additional acid–basecomponent that accounts for attractive hydrophobicinteractions and repulsive hydration effects, and thesecond model considers an additional exponentiallydecaying steric interaction between polymeric bru-shes in addition to the acid–base interactions. Thepredictions of those extended DLVO models agreedwell with the AFM experimental data, indicatingthat bacterial adhesion is significantly influenced bythe presence of extracellular structures. Herzberget 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 ofthe adhesion force was observed with the presenceof calcium ions, indicating a bridging effect betweenthe 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 ofRO membranes.

In SMFS, AFM tips are modified using mole-cules with specific biochemical activities to char-acterize molecular interactions and distributionsin biological systems (Muller and Dufrene 2008;Neuman and Nagy 2008). Francius et al. (2008)investigated the clinically important probioticbacterium Lactobacillus rhamnosus GG (LGG)wild-type and the CMPG5413 mutant whichwas impaired in adherence, biofilm formationand polysaccharide production. Cell surface

S. Liu and Y. Wang: Application of AFM in microbiology 67

polysaccharides were probed at the molecular levelto elucidate the differences. For this purpose, AFMtips 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 glycolchain. Two kinds of polysaccharide chains wereidentified on the cell surfaces of both strains: poly-saccharides rich in mannose (recognized by ConA-terminated tips) having moderate extensions,and polysaccharides rich in galactose (recognizedby PA-I-terminated tips) having much longer

extensions. The polysaccharide chains on LGGwild-type had much larger adhesion forces, longerrupture distances, and were more densely dis-tributed compared to those of the mutant. Thisstudy demonstrated the crucial role played by cellwall polysaccharides in determining nanoscale sur-face properties. Alsteens et al. (2008) studied twobrewing yeast strains, Saccharomyces carlsbergensisand S. cerevisiae with in situ AFM in three differentmodes. Although both strains are smooth andhomogeneous, an evident difference in cell wall

Fig 6. High-resolution deflection images (left) and adhesion force maps (right, scale bars: 100 nm; z range5 5 nN) recorded on asingle A. fumigatus spore during germination. The conidial surface had a crystalline rodlet look and was uniformly hydrophobicbefore the germination. After 2 h, both rodlet and amorphous regions were found to coexist (separated by dash line), andheterogeneous contrast was observed in the form of hydrophobic patches surrounded by a hydrophilic sea. Within 3 h, thecrystalline rodlet layer changed into a layer of amorphous material. The substantial reduction of adhesion contrast reflected adramatic decrease of hydrophobicity. Reprinted with permission from Dague et al. (2008).

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elasticity was revealed in nanomechanical measure-ments (Fig. 7(A–D)). In SMFS analysis with ConA-modified tips, major differences in polysaccharideproperties of the two strains were discovered. Poly-saccharides on S. cerevisiae surface were more ex-tended, suggesting that not only oligosaccharides butalso polypeptide chains of the mannoproteins werestretched (Fig. 7(E, F)). These findings at the nanoscale

and molecular level are useful to explain the differentaggregation properties of the two organisms.

Another remarkable application of SMFS is tostudy unfolding forces and mechanics of proteins.The pioneering work in measuring mechanicalproperties and unfolding force of individual titinmolecules 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-range5 3 MPa); (E), (F): three-dimensional reconstructed maps of polymer propertiesobtained by combining adhesion force values (expressed as false colors) and rupture distance (expressed as z level). Reprinted withpermission from Alsteens et al. (2008).

S. Liu and Y. Wang: Application of AFM in microbiology 69

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 adhesionprotein from the opportunistic pathogen Candidaalbicans. The forces required to unfold individualtandem repeats of the protein were determined to be150–250 pN. The unfolding probability increasedwith the number of tandem repeats and correlatedwith the level of cell adherence. The authors sug-gested that this modular elongation mechanismimparted 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 withone or more living cells that are used to measureinteractions toward other cells or substrates(Helenius et al. 2008). The force–distance curve re-corded when pulling the cell back from its objectivecan detect different unbinding events and discreteforce steps can be assigned to the rupture ofsingle cell adhesion molecules. Boks et al. (2008)investigated adhesion forces of four strains ofS. 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 hydrophobicdimethyldichlorosilane-coated glass was fast (lessthan 10 s), limited to a minor increase in adhesionforce, and likely governed by hydrophobic interac-tion. However, bond strengthening on hydrophilicglass occurred within 5–35 s to maximum adhesionforces of �1.970.7 nN and was concurrent with thedevelopment of multiple adhesion peaks uponretract. This was attributed to progressive formationof hydrogen bonds made possible by ongoingrearrangements of outer cell surface structures. As aconsequence, adhesion forces strengthened con-siderably more on hydrophilic glass than onhydrophobic glass. Poisson analysis of the multipleadhesion peaks allowed separation of contributionsof hydrogen bonding from other nonspecific inter-action forces and revealed a force contribution of�0.8 nN for hydrogen bonding and 10.3 nN forother nonspecific interaction forces. The time-de-pendent bacterial adhesion forces were comparablefor all four staphylococcal strains. Kang andElimelech (2009) developed a novel procedure forpreparing live bacterial cell probes for AFM using abioinspired polydopamine wet adhesive. Traditionaladhesion 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 quartzsurfaces measured with probes prepared through thenew 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 wasobserved.

Combining AFM with Other Analytical

Techniques

Although AFM is a powerful technique withmany advantages, this stand-alone tool has its ownlimitations. Combinations of AFM with othercomplementary techniques provide great opportu-nities for obtaining more comprehensive informa-tion from interesting samples, which furtherexpands AFM applications in microbiology (Floresand 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 AFMwith transmitted light optical microscopes, e.g.fluorescence lifetime imaging microscopy (FLIM)and total internal reflection fluorescence microscopy(TIRFM), can enable researchers to ‘‘look’’ at thesamples simultaneously.

In biological applications, organelles and mole-cular complexes can be selectively labeled withfluorescent dyes to make them visible. The combi-nation of AFM topography and fluorescence imagesprovides a unique tool for correlating organelles andchemical components with cell morphology. Micicet al. (2004) carried out an investigation on livingGram-negative S. oneidensis MR-1 cells with high-resolution AFM-tip-enhanced FLIM. A gene fusionof yellow fluorescent protein (YFP) to the methylaccepting 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 regionare shown in Figure 8(A–C), respectively. The AFMimage showed higher surface topography at bothends of the cells. Fluorescence images also showedhigher intensities at both ends of the cells, whichsuggests polar localization of the MCP-YFP fusionprotein. Therefore, a correlation between the sur-face protuberances in MR-1 cells and polar locali-zation of the MCP-YFP fusion protein could beconcluded based on the combination of the AFMand fluorescence images.

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TIRFM uses evanescent waves to selectivelyilluminate and excite fluorophores in a restrictedregion of the specimen immediately below the totalreflection interface (typically a few tens of nan-ometers). As a consequence, a high-resolutionfluorescence image of a sample surface can be ob-tained. Shaw et al. (2006, 2008) investigated how acationic antimicrobial peptide, indolicidin, inter-acted with model membranes, mixed zwitterionicplanar lipid bilayers, with AFM-confocal-TIRFM.The results indicated that the peptide rearranged themodel membranes in a concentration- and lipid-dependent manner. At low peptide concentration, itappeared to reduce the interfacial line tension at thedomain boundary between the liquid-ordered andliquid-disordered domains. Peptide-induced mem-brane remodeling only occurred under high peptideconcentration. These works demonstrated that cor-related AFM-confocal-TIRFM imaging is an at-tractive approach for tracking preferentiallocalization of fluorescent lipid probes and wellsuited 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) isa label-free nanoscale chemical characterizationtechnique. It uses a sharp metal tip (e.g. Ag or Au).When the tip is sufficiently close (ca. 1 nm) to thesample, the near field can optically excite nearbymolecules on the sample surface, and thus, localRaman spectra can be obtained. Combining AFMwith TERS should be a promising way to improvechemical recognition ability for AFM. Schmidet al. (2008) employed a well-defined model system

consisting of calcium alginate fibers to evaluate thefeasibility of the combination of AFM and TERS inbiological systems. Alginates are stabilizing com-pounds in the extracellular polymeric substances ofcertain biofilms. The mainly guluronic acid-richparts of alginate can interact with Ca21 ions andcross-link different alginate strands resulting in athree dimensional network-like structure, which is agood model system for investigation of microbialaggregates. The investigation of calcium alginatefibers showed that Raman frequencies in TERSspectra of biopolymers do not necessarily resembleband positions in the normal Raman spectrum ofthe bulk material. Additionally, analyte decom-position due to laser heating and carbon con-tamination were observed. Fortunately, strategiesfor spectra correction, choice of appropriatereference samples and data interpretation werepresented by the authors. With this approach,characteristic frequency ranges and specific markerbands can be found for biological macromolecules,which can be employed for their identification incomplex environments.

Conclusions

AFM is a versatile tool that images cellular sur-faces at high resolution and also measures funda-mental interactions giving cells their characteristicstructure–function relationship (Muller et al. 2009).Although a topography measurement of air-driedsamples is still the most common application ofAFM in microbiology, morphology probing onliving cells will be more frequently employed in thefuture given improvements in the instrument andoperators’ skill. Besides imaging, force–distancecurves generated using AFM show great potentialfor elucidating microbial interactions and physicalproperties. Through modification of the AFM tip, itis possible to investigate many forms of interactions

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 andlifetime 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).

S. Liu and Y. Wang: Application of AFM in microbiology 71

over scales ranging from molecules to cells. Theapplications of CFM, SMFS and SCFS have beendramatically expanded in the last few years and willremain a focus of investigation in the near future.Combinations of AFM with other complementarytechniques are being considered by more and moreresearchers and developments in this area will opennew ways to look at biological samples. In sum-mary, AFM-based measurements will continue toplay an important role in future microbiologicalresearch, and improve our understanding of thestructures and properties of microbial cell surfacesand the mechanisms involved in microbial behaviorat the molecular level.

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