Download In situ high-resolution atomic force microscope imaging of biological

In situ high-resolution atomic force microscope imaging
of biological surfaces
I. Yu. Sokolova
Department of Physics, University of Toronto, Toronto M5S 1A7, Canada
M. Firtelb)
Department of Microbiology, University of Toronto, Toronto M5S 1A7, Canada
G. S. Hendersonc)
Department of Geology, University of Toronto, Toronto M5S 1A7, Canada
~Received 29 September 1995; accepted 18 March 1996!
In situ high-resolution atomic force microscope imaging of biological surfaces was performed on
cells with relatively rigid surfaces ~e.g., bacteria!. The surface of Lactobacillus helveticus ~a
rod-shaped bacterium! was investigated before and after exposure to LiCl, a denaturant. Image
details were stable both at variant force loads and under different scan directions. From images of
the oblique lattice structure ~i.e., S layer of L. helveticus!, it was estimated that the lateral resolution
of the images was up to 2 nm. This resolution can be explained by assuming that there is an apex
with a curvature of radius of ;10 nm near the end of the tip. Modelling of this geometry indicates
that such a tip configuration is particularly suitable for in situ high-resolution imaging of relatively
soft objects covered by a rigid shell ~membrane!. © 1996 American Vacuum Society.
I. INTRODUCTION
Atomic force microscopy ~AFM! is a novel technique
based on measuring intermolecular forces between a sharpened tip and the surface of a sample.1– 4 Like any other microscopy technique an important characteristic of the instrument is the spatial resolution attainable when imaging
objects of interest. For sufficiently rigid surfaces, the vertical
resolution of an AFM is primarily determined by the sensitivity of the height/deflection detector, whereas lateral resolution is strongly dependent on tip geometry ~see, e.g., Refs.
3, 5, and 6!. A consequence of this is that the lateral resolution attainable is usually less than the vertical resolution and
is therefore the limiting factor for imaging. However, as we
shall demonstrate in this work, for a rather soft material, the
vertical resolution may be restricted by the elasticity of the
surface, and consequently, the vertical resolution can also be
a limiting factor.
Currently, the AFM is able to achieve atomic horizontal
resolution on nonbiological samples, while studies of biological samples have reported a lateral resolution of 1–3
nm.7–17 However, this has only been achieved for either molecules, or small complexes, such as cholera toxin
B-oligomer, that have been attached to relatively rigid substrates.
In the present work, we present the results of a study in
which the surface of Lactobacillus helveticus ~a rod-shaped
bacterium! has been imaged in situ. @It should be noted that
the term ‘‘in situ’’ is used here for rehydrated bacteria after
1–2 min of drying. We use ‘‘in situ’’ because it has been
found that such drying is not dangerous for the bacteria ~cf.,
Ref. 18! even after long drying periods. In addition, the baca!
Electronic mail: [email protected]
Deceased July 6, 1995.
c!
Electronic mail: [email protected]
b!
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teria are normally alive after the drying process before and
after chemical treatment by a denaturant to remove the outer,
so-called S layer, of the cell.# The lateral resolution of the
images is approximately 2 nm and a model is proposed to
explain the resolution achieved. The S layer of Bacillus coagulants E38-66, and Bacillus sphaericus CCM2177 has
been studied previously by AFM.1 However, in that study the
S-layer protein was extracted and deposited onto a rigid support prior to imaging. Lateral resolution was not reported but
can be estimated from the images in Ref. 14 as 5–10 nm.
II. MATERIALS AND METHODS
A. Atomic force microscope
All the images were obtained using a Digital Instruments
NanoScope III AFM operating in contact mode. The A1B
signal of the feedback was about 3 V while the A–B signal
was set at around 21 V and the setpoint at 0 V ~repulsion
force ;1029 N!. Standard silicon nitride integrated pyramidal tips with an estimated radius of curvature of ;100 nm, or
chemically etched silicon supertips with a radius of ;25 nm,
were fixed on 200 mm ‘‘wide leg’’ cantilevers ~spring constant k;0.06 N/m!. The radii of curvature were estimated by
scanning of a rectangular step on a mica surface. The supertip was used for scanning in air while a standard tip was used
for scans in both water and air. The D scan head ~maximum
scan area is 12.5312.5 mm2, z sensitivity is 9 nm/V! was
employed throughout the study. All the images were reproducible under a variety of instrument conditions including
variable scan direction and contact force. The spacing of the
oblique lattice structure ~S layer! of L. helveticus17–19 was
used to calibrate the lateral resolution of the microscope.
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Sokolov, Firtel, and Henderson: AFM imaging of biological surfaces
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B. Specimen preparation
The specimen was deposited on an atomically flat surface
of muscovite ~mica!, which was fixed on the a sample stub
by double sided sticky tape. In air scans were performed on
untreated muscovite, whereas the muscovite was precoated
with a film of media to promote attachment of bacteria when
scanning in water.
The precoated muscovite surfaces were prepared by maintaining a droplet of MRS broth on the mica surface for several hours to overnight in a humidified chamber kept at 4 °C.
This resulted in deposition of a thin film on the mica surface
that was found to promote attachment of cells. The cells
were allowed to sorb to the precoated mica for several hours
then were washed once in pure water and dried at room
temperature for several minutes. The cells were then rehydrated in the fluid chamber mounted inside the AFM and
imaged while they were immersed under water. For imaging
in air, cells in suspension ~OD650 ;1.0! were sorbed for
several minutes on untreated muscovite. Excess suspension
was removed leaving a thin film which was allowed to dry.
Nonadhered cells and debris were removed by washing the
surface with distilled water. After blotting and air drying,
enough cells remained on the surface to be detected within
several scans at maximum range.
C. LiCl treatment and cell preparation
Cells were collected by centrifugation ~12 5003g; 10
min!, washed once in distilled water, and resuspended in 5 M
LiCl ~10-15 mg of moist pellet ml-1 LiCl! for 10 min at 0 °C,
followed by centrifugation at 12 5003g for 20 min. As
shown in Refs. 17–19 such a treatment removes the S layer
of L. helveticus. The treated pellet was washed once in distilled water and resuspended in prewarmed ~43 °C! MRS
broth medium to an OD650 of 0.05 and incubated with shaking at 43 °C in a CO2 atm.
Cells were collected and imaged before treatment, immediately on resuspension into broth ~time zero!, and at the end
of S layer regeneration ~time zero19.5 h!.
III. EXPERIMENTAL RESULTS
A high-resolution image of L. helveticus surface before
the treatment is shown in Fig. 1~b! and Fig. 1~a! shows a
general view of a bacterium. The area marked by a square is
zoomed in Fig. 1~b!. We did not see any significant difference between images collected in air or in water, however,
imaging in fluid reduces instrumental noise producing higher
quality images. The geometrical size of details on the cell
surface indicate that the lateral resolution is about 2 nm.
In this article we estimate the lateral resolution as the
radius of the smallest feature of the surface that can be reliably distinguished. This may not seem to be a rigorous definition but we find it to be quite useful when examining an
unknown surface. This is because the normal definition of
resolution is related to the degree of ‘‘washing out’’ of some
sort of topographic pattern. However, we do not know the
real pattern of our surface better than ;5 nm @from transmisJVST A - Vacuum, Surfaces, and Films
FIG. 1. ~a! General view of untreated bacteria. Zoom area is indicated by a
square. ~b! Image of untreated bacteria surface. Maximum verical height
change is 0.4 nm.
sion electron microscopy ~TEM!# and the true resolution of
the images may be better than the stated value of 2 nm.
As noted previously, the image details were not affected
by different scan angles or contact forces, however, a nontrivial dependence on the scan rate was found. For scan rates
between 4 –5 and 20– 40 Hz, and with integral and proportional gain parameters around 4 –5, we observed a series of
image artifacts that occur in the images as parallel lines with
a periodicity from ;2 to 20 nm. The dependence of the
artifacts on the scan rate is best explained by frictional effects. An analogy would be the movement of a wet finger
~bacterium surface! on the surface of glass ~tip!, one can see
that the motion is smooth provided the speed of the finger is
slow. Increasing the speed, one can feel that finger moves by
fits and starts while a further increase in speed reverts back
to smooth motion.
Figure 2 shows an image of the bacteria in water after
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Sokolov, Firtel, and Henderson: AFM imaging of biological surfaces
FIG. 2. ~a! General view of bacteria just after LiCl treatment. Zoom area is
indicated. ~b! Image of the bacteria surface just after LiCl treatment. Maximum verical height change is 0.5 nm.
treatment by LiCl. Figure 2~a! shows a general view as did
Fig. 1~a!. Some damage is visible even in this scale. The
square area that was zoomed is indicated in Fig. 2~b! is indicated. As one can see in Fig. 2~b!, the character of the
surface has been changed dramatically. Instead of the regular
S-layer pattern, ‘‘holes’’ are observed. These holes have diameters of up to 10 nm and a surface density of ~862! holes/
1000 nm2. The estimated resolution for this image is ;5 nm
~it should be noted that we were not able to obtain good
high-resolution images in air!.
A general view of the bacteria after 9.5 h is shown in Fig.
3~a!. One can see both relatively ‘‘smooth’’ bacteria and
rather ‘‘rough’’ ones. It is worth noting that the regenerated S
layer was not been observed on the rough bacteria. It should
be noted that Fig. 3~a! was done in air. After adding water,
the difference in roughnesses of the bacteria was not so obvious.
High-resolution imaging of a square area in Fig. 3~a! is
J. Vac. Sci. Technol. A, Vol. 14, No. 3, May/Jun 1996
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FIG. 3. ~a! General view of bacteria 9.5 h after LiCl treatment. Zoom area is
indicated. ~b! Image of the bacteria surface 9.5 hr after LiCl treatment.
Maximum verical height change is 0.5 nm.
presented in Fig. 3~b!. It is known from standard TEM
studies17,19 that S layers removed by LiCl are regenerated
after 9 h. This pattern of regeneration was also observed in
this study @Fig. 3~b!#, indicating that the features observed in
our images are indeed real and not artifacts of the technique.
However, as one can see from Fig. 3~b!, the recovery of the
S layer does not seem to be completed after 9.5 h @cf. Fig.
1~b!#. The white lines in the image are presumably caused by
highly localized sticking particles ~molecules!. This may
suggest the presence of a significant number of unreacted
chemical bonds on the new surface. Similarly, large corrugations of the surface with increasing contact force ~unshown!
are indicators of a softer bacterial surface after regeneration.
Furthermore, the regenerated S layer appears to be stickier
than the one prior to treatment. This manifests itself as increased noise in the images due to an increase in highly
localized interactions between the tip and the surface. This
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Sokolov, Firtel, and Henderson: AFM imaging of biological surfaces
FIG. 4. ~a! Model of microapex at the top of the AFM tip. ~b! The microapex
over a model surface with a characteristic scale of ;3 nm.
noise is probably the main reason why the resolution in Fig.
3 of ;4 –5 nm is lower than in Fig. 1.
IV. THEORETICAL MODEL FOR HIGH-RESOLUTION
IN SITU IMAGING
The lateral resolution of 2–3 nm obtained for our images
is not possible if the radius of curvature of the conventional
pyramidal tips used to image the bacteria is truly 50–100
nm. This was estimated by scanning a mica steps. Such tips
would limit the attainable resolution to 10– 40 nm.6,20 In addition, biological materials are generally soft and, as a result
of surface deformation, there is ‘‘enveloping’’ of the AFM tip
by the biosample. This increases the tip–sample contact area,
producing further deterioration of the resolution.
In order to achieve the observed resolution, we must assume that there is a microapex on the tip @Fig. 4~a!#. A similar model was proposed by Shao and Yang21 for a homogeneous material ~with a Youngs modulus not less that 108
N/m2! on a planar surface. Here, we consider a round surface
of relatively soft bulk material surrounded by a much more
rigid ~than bulk! membrane.
Figure 4~b! shows the configuration of the apex, with radius R 5 10 nm, over a model surface with features of ;3 nm
in size. Because our model considers the a rigid membrane
surrounding a soft bulk, we can assume that deformation of
the membrane should be negligible. This is consistent with
the value of the Young’s modulus for the S layer below. We
can therefore approximate the tip surface geometry with the
simple schematic shown in Fig. 4~b!. The height of the microapex must be greater than the height of the S layer
~;0.3–0.6 nm! or the tip would not be able to image between topographic ‘‘highs’’ on the sample surface. Based on
JVST A - Vacuum, Surfaces, and Films
677
the experimental data for the untreated surface ~showed in
Fig. 1!, we have to expect both a lateral resolution of 2 nm
and a visible vertical height of 0.3–0.6 nm for the S layer. As
one can see now from Fig. 4~b!, the apex radius R<10 nm
and height >0.6 nm are enough to produce both the aforementioned lateral resolution and the observed height of the S
layer.
Let us now consider the problem of vertical resolution. As
was noted previously, the vertical resolution is usually determined by the sensitivity of the instrumental height/deflection
detection system. However, for the sufficiently soft surface,
the vertical resolution may be dependent upon the elasticity
of surface being imaged. This is because an elastic surface
could be deformed by the tip. This is a nontrivial problem.
For example, in this study, the tip could deform the S layer of
bacteria by ‘‘pushing’’ relatively high surficial features into
the bulk of the cell so that no change of height is observed in
the image.
The height variation of the S layer observed in Fig. 1 is
;0.5 nm. The NanoScope III system is sensitive to a change
in vertical force ( d F! of better than ;1022 nN. This means
that we cannot observe any structure on the surface of the
bacteria that does not produce a change in d F of less than
this magnitude. We can estimate the magnitude of the surface
relief required to generate such a change in force for the
imaging conditions we have proposed. We now consider a
very soft bacterium bulk ~protoplasm! surrounded by a rigid
S-layer sheath. Assuming a spherical membrane with a pointlike force ~see Ref. 22!, then the deformation, H, is given by
H'
d FR bact
Eh 2
,
where Rbact is the radius of the shell ~the bacterium radius!, E
is the Young’s modulus for the S-layer sheath, and h is the
thickness of the sheath.
The pointlike force approximation, however, is only valid
provided R bact /r 2 @ 1 is true,16 where r is the radius of the
area of force application. For R bact 5 0.5 mm and h510 nm, it
means h ! 70 nm, which is true for our geometry ~cf. Fig. 4!.
Assuming a value of E ; 1010 N/m2 for L. helveticus
based on the Young’s modulus ( ; (2 6 1) 3 1010 N/m2! for
the S-layer sheath of archeobacterium Methanospirillum
hungati,23 then we can determine that
H'1022 nm
S
R bact
0.5 mm
DS
1010 N/m2
E
DS
D
10 nm 2
.
h
This indicates that our AFM is able to detect variations in
S-layer height of 0.5 nm with an error of ;2%, which is
more than enough to produce the image of Fig. 1. In general,
one can see from this formula that diminishing of rigidity ~by
either increasing R or by decreasing E or h! leads to decreasing the vertical resolution. This implies that cell rigidity may
be a crucial factor in determining the vertical resolution
when imaging biosurfaces.
We have not considered the geometry of the membrane
nor taken into account frictional forces across the bacterium
surface; however, nonideality of the tip is sufficient to ex-
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Sokolov, Firtel, and Henderson: AFM imaging of biological surfaces
plain the resolution observed. This simple approximation of
a microapex at the AFM tip can explain the resolution obseved.
V. DISCUSSION
We have presented high-resolution in situ AFM images of
the rod-shaped bacterium Lactobacillus helveticus prior to
and after chemical treatment by LiCl. Images of bacteria
prior to treatment indicate that a lateral resolution of 2 nm
was achieved. This resolution can be explained by the presence of a microapex on the AFM tip. Resolution was diminished after LiCl treatment and after regeneration of the S
layer and is probably a consequence of surface softening. It
is also shown that, in contrast to the imaging of rigid
samples, the vertical resolution may be the restrictive factor
when imaging soft biosamples.
The tip configuration considered above is probably better
than the chemically etched silicon ‘‘supertips’’ for in situ
high-resolution imaging of biosurfaces. If we compare a supertip with a curvature radius of ;10 nm and a normal tip
with a radius of ;100 nm and apex of ;10 nm, one may
expect the same lateral resolution for relatively flat surfaces.
However, for a surface with significant vertical relief the
supertip is at a disadvantage when compared to our proposed
modified tip. A supertip is relatively sharp and will produce
much higher lateral ~friction! forces than our proposed geometry. It is likely that a supertip will tend to push bacteria
around the surface and/or ‘‘shred’’ the cells. In general, we
were unable to obtain good images of bacteria in water when
using a supertip. This was because we did indeed tend to
push the bacteria around the surface or in the rare cases
where a bacterium was located it was quickly destroyed after
a few scans. The modified tip imparts less pressure on the
bacterial surface ~up to 100 times less! and if the height of
the microapex is not greater than the thickness of the cell
membrane, then it will be unable to penetrate the cell and
will consequently destroy the bacteria. This suggests that our
proposed tip geometry is very useful for imaging soft materials covered with a relatively rigid membrane ~bacteria!.
ACKNOWLEDGMENTS
The authors are grateful to P. Sivarajah for the preparation
of L. helveticus for this work and Professor Terry Beveridge
J. Vac. Sci. Technol. A, Vol. 14, No. 3, May/Jun 1996
678
for helpful discussions. They also appreciate the support and
encouragement of J. Campbell, J. Fawcett, and P. Sadowski.
This study was supported by start-up funds from the Department of Microbiology, University of Toronto to two of the
authors ~M.F. and I.Yu.S.! and NSERC research and equipment grants to the third ~G.S.H.! Co-author Max Firtel
passed away suddenly prior to completion of this manuscript.
His intellect, advice, wit, and friendship will be sorely
missed by us both.
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