procedure for AFM of native microbial
cells and presents a series of examples
to highlight the various applications ofthe technique in this field. All of thedata in this application note wasobtained using a Digital Instruments
MultiMode® 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 for
probing living (non-fixed) animal cells
was recognized quite early2, attempts
to perform such measurements on
microbial cells have been morelimited, due in part to the difficulties
associated with the sample
preparation. As opposed to animalcells, microbial cells have a welldefined shape and have no tendency
to 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 as
microbial cells. To circumvent theseproblems, a convenient immobilization
procedure is to trap the cells in a
porous filter membrane3,4.
As shown in Figure 1, a concentrated
cell suspension is gently sucked
through a polymer membrane witha pore size slightly smaller than thecell size. After cutting the filter(1cm x 1cm), the lower part is quickly
dried on a sheet of tissue and thespecimen is then attached to thesample holder using a small pieceof adhesive tape. The main advantage

2of this procedure is that it does not
involve chemical treatments which
could cause rearrangement/denaturation of the surface molecules.
Examples of AFM height images
showing single cells trapped in apore of the membrane are shown inFigures 2 and 3, for the fungus
Aspergillus oryzae and the bacterium
Lactococcus lactis, respectively. The
cells can be imaged in an aqueous
solution repeatedly without celldetachment or surface damage.

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

Applications
Imaging
AFM can provide high-resolution
images of the surface structure (cellwall layers, appendages) of microbial
cells5. Both height and deflection
images are often useful because theyyield complementary information.
Height images (Figures 2 and 3)
provide quantitative heightmeasurements, thus allowing an
accurate measurement of celldimensions and of the surface
roughness. Deflection images do notreflect true height variations, but
because they are more sensitive tofine surface details they are usefulfor revealing the surface ultrastructureof curved, rough samples such as
cells. An example of high-resolution
deflection image is shown in Figure 4
for the surface of A. oryzae spores inthe dormant state. The surface iscovered with patterns of nanoscalerodlets, presumably proteins, which
are assembled in parallel to form
supramolecular fascicles6. Althoughsuch structures were previously
observed by electron microscopy,
they can now be visualized directly
in aqueous solution.

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

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

Appendages (fimbriae, fibrils, flagella)
is another class of surface structuresoften found on bacterial cells. Hereagain, AFM can be used to image
these structures directly in the nativestate. For instance, fibrils about 200nm
in length could be detected on thesurface of the bacterium Streptococcus
salivarius HB7. Note, however, that
due to the soft and fragile nature of
the fibrils, the image quality is not
excellent and the contrast mechanism
unclear, emphasizing the need to use
alternative immobilization procedures
and imaging modes.
Of particular interest for microbiologists
is the ability to study physiologicalprocesses such as cell growth andgermination. Figure 3 shows two
dividing daughter cells, indicating that
AFM could be used to follow division
processes in real time. Anotherexample is the germination of fungal
spores, a process which is important
both in natural and biotechnologicalsituations. Figures 5 shows a deflection
image of the surface of A. oryzae aftera 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 attributed
to polysaccharides6.
Force Measurements
AFM is actually much more than a
topographic imaging tool in that it
also enables physical properties to bemeasured. Force-distance curves canbe used to probe long-range surface
forces (e.g., van der Waals andelectrostatic forces) and to map
adhesion forces on microbial cellsurfaces, thereby providing new insight
into the molecular basis of biologicalevents such as cell adhesion. As an
example, Figure 6 shows a set ofdata obtained for the surface of agerminating spore of the fungus
Phanerochaete chrysosporium4,5. The
heterogeneous surface morphology(Figure 6a) is directly correlated withdifferences in adhesion forces asrevealed by retraction force curves.
While no adhesion forces are sensed
between the probe and the granularmaterial (top curve, Figure 6c), strong
adhesion forces of 9 ± 2 nN
magnitude are measured on thesmooth zone (bottom curve, Figure 6d).
Force-volume imaging, which consists
in recording arrays (typically, 64 x 64)
of force curves in the x, y plane,
provides spatially resolved force
measurements on biological samples8.
The power of this approach in
microbiology is illustrated in Figure 6.

a.
c.

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

3Figure 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. The
smoother 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.

Differences in adhesion forces acrossthe spore surface yield a highly
contrasted adhesion map (Figure 6b),which is correlated with theheterogeneous surface morphology(Figure 6a). The measured adhesion
forces may be of great biologicalsignificance in that they may be
responsible for cell aggregation
observed during germination.
In addition to measuring non-specificforces, the AFM probe can befunctionalized with various molecules
to measure specific interactions.
For instance, OH- and CH3terminated probes were used toquantitatively map the surfacehydrophobicity (surface energy) ofP. chrysosporium spores9.
With the AFM probe, one can also
push locally onto cell wall components
and surface appendages to measure
their mechanical properties
quantitatively. These properties play
an important role in controlling events
such as cell growth, cell division andcell adhesion. Force-distance curves,
recorded in buffer solution, were used
to determine the effective
compressibility of the cell wall of themagnetic bacterium Magnetospirillum
gryphiswaldense10, and to measure thesurface softness of the fibril layer at thesurface of the bacterium Streptococcus
salivarius HB 7.
Finally, AFM can be used to pull onmacromolecules to learn about theirelastic properties. Single molecule
force spectroscopy experiments
have provided new insight into thenanomechanical properties of single
DNA, protein and polysaccharide
molecules11. Stretching single molecules
directly on living cells remains very
challenging due to the complex anddynamic nature of the surface.
However, data indicates that this goal
could soon be achieved. As seen in
Figure 7, retraction force curves
recorded on germinating spores ofA. oryzae showed attractive forcesof 400 ± 100pN magnitude, alongwith characteristic elongation forces

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

4and rupture lengths of up to 500nm.
Elongation forces were well-fitted withan extended freely jointed chain (FJC+)
model from statistical mechanics, using
fitting parameters that were consistent
with the stretching of individual
dextran polysaccharide molecules.
This observation, together with the
topographic information (Figure 5)
and with biochemical data, suggestthat the measured forces may reflect
the stretching of long, flexible cellsurface polysaccharides.

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

5