procedure for AFM of native microbialcells and presents a series of examplesto highlight the various applications ofthe technique in this field. All of thedata in this application note wasobtained using a Digital InstrumentsMultiMode® atomic force microscope(AFM) equipped with a fluid cell.

Sample preparation

Cells were among the first biologicalsamples to be investigated by AFM1.While the potential of the instrument forprobing living (non-fixed) animal cellswas recognized quite early2, attemptsto perform such measurements onmicrobial cells have been morelimited, due in part to the difficultiesassociated with the samplepreparation. As opposed to animalcells, microbial cells have a well-defined shape and have no tendencyto spread over substrates. As a result,the contact area between cells andsubstrate is very small, often leading tocell detachment by the scanningprobe. A second issue is the verticalmotion of the sample (or probe),typically limited to a few micrometers,which makes it difficult to image thesurface of large objects such asmicrobial cells. To circumvent theseproblems, a convenient immobilizationprocedure is to trap the cells in aporous filter membrane3,4.

As shown in Figure 1, a concentratedcell suspension is gently suckedthrough a polymer membrane with a pore size slightly smaller than the cell size. After cutting the filter (1cm x 1cm), the lower part is quicklydried on a sheet of tissue and thespecimen is then attached to thesample holder using a small piece of adhesive tape. The main advantage

Figure 3. Topography of two dividingbacterial cells (Lactococcus lactis). 5µm x 5µm.

of this procedure is that it does notinvolve chemical treatments whichcould cause rearrangement/denaturation of the surface molecules.Examples of AFM height imagesshowing single cells trapped in a pore of the membrane are shown inFigures 2 and 3, for the fungusAspergillus oryzae and the bacteriumLactococcus lactis, respectively. Thecells can be imaged in an aqueoussolution repeatedly without celldetachment or surface damage.

Applications

ImagingAFM can provide high-resolutionimages of the surface structure (cellwall layers, appendages) of microbialcells5. Both height and deflectionimages are often useful because theyyield complementary information.Height images (Figures 2 and 3)provide quantitative heightmeasurements, thus allowing anaccurate measurement of celldimensions and of the surfaceroughness. Deflection images do notreflect true height variations, butbecause they are more sensitive to fine surface details they are useful for revealing the surface ultrastructureof curved, rough samples such ascells. An example of high-resolutiondeflection image is shown in Figure 4for the surface of A. oryzae spores inthe dormant state. The surface iscovered with patterns of nanoscalerodlets, presumably proteins, which are assembled in parallel to formsupramolecular fascicles6. Althoughsuch structures were previouslyobserved by electron microscopy, they can now be visualized directly in aqueous solution.

Figure 4. Visualizing cell surfacenanostructures. High-resolution deflectionimage in aqueous solution of the surface of a dormant spore (A. oryzae). The surface iscovered with well-ordered nanostructures,called ”rodlets,” that are 10nm in diameterand assembled in parallel to form fasciclesinterlaced in different orientations. 750nm x 750nm.

Figure 2. 3D topography showingfungal spore (Aspergillus oryzae)trapped into a pore of the membrane.10µm x 10µm.

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data obtained for the surface of agerminating spore of the fungusPhanerochaete chrysosporium4,5. Theheterogeneous surface morphology(Figure 6a) is directly correlated withdifferences in adhesion forces asrevealed by retraction force curves.While no adhesion forces are sensedbetween the probe and the granularmaterial (top curve, Figure 6c), strongadhesion forces of 9 ± 2 nNmagnitude are measured on thesmooth zone (bottom curve, Figure 6d).

Force-volume imaging, which consistsin recording arrays (typically, 64 x 64)of force curves in the x, y plane,provides spatially resolved forcemeasurements on biological samples8.The power of this approach inmicrobiology is illustrated in Figure 6.

Appendages (fimbriae, fibrils, flagella)is another class of surface structuresoften found on bacterial cells. Hereagain, AFM can be used to imagethese structures directly in the nativestate. For instance, fibrils about 200nmin length could be detected on thesurface of the bacterium Streptococcussalivarius HB7. Note, however, thatdue to the soft and fragile nature of the fibrils, the image quality is notexcellent and the contrast mechanismunclear, emphasizing the need to usealternative immobilization proceduresand imaging modes.

Of particular interest for microbiologistsis the ability to study physiologicalprocesses such as cell growth andgermination. Figure 3 shows twodividing daughter cells, indicating thatAFM could be used to follow divisionprocesses in real time. Anotherexample is the germination of fungalspores, a process which is importantboth in natural and biotechnologicalsituations. Figures 5 shows a deflectionimage of the surface of A. oryzae after

a few hours of germination. Clearly,spore germination causes dramaticchanges of the surface structure: the crystalline rodlet layer (Figure 4)changes into a layer of soft,deformable material attributedto polysaccharides6.

Force MeasurementsAFM is actually much more than atopographic imaging tool in that it also enables physical properties to bemeasured. Force-distance curves canbe used to probe long-range surfaceforces (e.g., van der Waals andelectrostatic forces) and to mapadhesion forces on microbial cellsurfaces, thereby providing new insightinto the molecular basis of biologicalevents such as cell adhesion. As anexample, Figure 6 shows a set of

Figure 5. Probing physiological changes.Deflection image showing the dramaticchange of the spore surface morphology upon germination. 2µm x 2µm.

Figure 6. Spatially resolved force measurements using force-volume imaging. Topographic image(a) and adhesion map (b) acquired on the same area of a germinating spore of P. chrysosporium.The adhesion map is obtained by recording 64 x 64 force-distance curves, calculating theadhesion force for each force curve and displaying adhesion force values as grey levels. Thesmoother area in the center of the image shows strong adhesion forces, which are thought to play acentral role in spore aggregation. Representative force-distance curves are shown on the right (c).2µm x 2µm scan.

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a.

b.

c.

d.

Figure 7. Stretching cell surface macromolecules. Typical force-distance curve recordedon germinating spores of A. oryzae. Retraction curves are well-fitted with an extendedFJC model (blue line), with parameters consistent with values reported for the elasticdeformation of single dextran and amylose polysaccharides.

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Differences in adhesion forces acrossthe spore surface yield a highlycontrasted adhesion map (Figure 6b),which is correlated with theheterogeneous surface morphology(Figure 6a). The measured adhesionforces may be of great biologicalsignificance in that they may beresponsible for cell aggregationobserved during germination.

In addition to measuring non-specificforces, the AFM probe can befunctionalized with various moleculesto measure specific interactions. For instance, OH- and CH3-terminated probes were used toquantitatively map the surfacehydrophobicity (surface energy) of P. chrysosporium spores9.

With the AFM probe, one can alsopush locally onto cell wall componentsand surface appendages to measuretheir mechanical propertiesquantitatively. These properties play an important role in controlling eventssuch as cell growth, cell division and

cell adhesion. Force-distance curves,recorded in buffer solution, were usedto determine the effectivecompressibility of the cell wall of themagnetic bacterium Magnetospirillumgryphiswaldense10, and to measure thesurface softness of the fibril layer at thesurface of the bacterium Streptococcussalivarius HB 7.

Finally, AFM can be used to pull onmacromolecules to learn about theirelastic properties. Single moleculeforce spectroscopy experiments have provided new insight into thenanomechanical properties of singleDNA, protein and polysaccharidemolecules11. Stretching single moleculesdirectly on living cells remains verychallenging due to the complex anddynamic nature of the surface.However, data indicates that this goalcould soon be achieved. As seen inFigure 7, retraction force curvesrecorded on germinating spores of A. oryzae showed attractive forces of 400 ± 100pN magnitude, along with characteristic elongation forces

and rupture lengths of up to 500nm.Elongation forces were well-fitted withan extended freely jointed chain (FJC+)model from statistical mechanics, usingfitting parameters that were consistentwith the stretching of individual dextran polysaccharide molecules. This observation, together with thetopographic information (Figure 5) and with biochemical data, suggestthat the measured forces may reflectthe stretching of long, flexible cellsurface polysaccharides.

SummaryAFM provides exciting possibilities forprobing the structural and physicalproperties of living microbial cells.Using topographic imaging, cellsurface nanostructures (rodlets,appendages) can be directlyvisualized and the changes of cellsurface morphology occurring duringphysiological processes (germination,division) can be determined. Force-distance curves providecomplementary information on surfaceforces, adhesion and nanomechanics,yielding new insight into themechanisms of biological events suchas cell adhesion and aggregation.Improvements in sample preparationtechniques, instrumentation andrecording conditions may bringsubnanometer resolution to these livingcells for monitoring molecularconformational changes, as is alreadythe case with reconstituted microbialsurface layers12.

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