Ultramicroscopy 50 (1993) 157-170

North-Holland
ultramicroscoov
I .
Effect of probe force on the resolution
of atomic force microscopy of DNA
Jie Yang and Zhifeng Shao *
Bio-SPM Laboratory and Department of Physiology, University of Krginia, Box 449, Charlottesville, VA 22908, USA
Received 15 December 1992
Experimental results are presented to show that the adhesion force is the single most important limiting factor in
high-resolution atomic force microscopy of DNA in air, prepared by the cytochrome-C-assisted spreading method. It is also
shown that humidity plays a minor role in the control of probe force. Using a pure carbon film as the substrate to clean the
APM tip prior to imaging, it is demonstrated that 4-6 nm resolution on DNA can be routinely obtained by the atomic force
microscope with commercial Si,N, pyramid cantilevers. We also show that in organic solvents a resolution of up to 3 nm can
be obtained under optimal conditions
1. Introduction
The atomic force microscope (AFM) has been
applied to various biological materials in air and
in fluid. Despite the soft nature of these speci-
mens and the relatively large probe force re-
quired with the instruments available today, some
remarkable results have been published, such as
AFM imaging of native membrane fragments [ 1,2],
synthetic lipid bilayers [3-81, purified membrane
proteins [61, and both double-stranded and
single-stranded DNA [9-171. For imaging DNA,
several specimen preparatory methods have been
developed [9-153 that yielded highly reproducible
results with the best resolution comparable or
exceeding those achieved by electron microscopy
of stained or heavy-metal-shadowed specimens
[18,191. Our preparatory method used the con-
ventional cytochrome-C-assisted spreading of
DNA on carbon-coated mica [20]. With this
method without further fixation or staining or
shadowing, the DNA molecules can be directly
visualized by AFM either in air or in some or-
ganic solvents, with a consistent lateral resolution
of 4-6 nm [9] (fig. 1). A molecular width of 3 nm
* To whom correspondence should be addressed.
was observable in fluids, such as isopropanol and
n-butanol. The specimens prepared by this
method are extremely stable that, when stored in
air for over 6 months, the same resolution can
still be achieved without apparent sign of degra-
dation. Repeated scanning with controlled probe
force usually does not damage the specimen.
Other methods have not used the protein-assisted
DNA spreading [lo-151. To facilitate DNA adhe-
sion to the substrate, mica was treated by various
chemicals, such as 3-aminopropyltriethoxy silane
[15,17] and magnesium acetate [11,12,16]. With
these methods, the specimen can also be imaged
in air or in fluid. Using a micro-fabricated su-
pertip 3 nm resolution was achievable in some
organic solvents [12,161. With the silane-treated
mica, DNA was shown to adhere rather strongly
to the substrate [15,171. Furthermore, the silane-
treated method also achieved DNA imaging in
water [17]. These recent developments firmly es-
tablished the validity of AFM results on imaging
DNA molecules, which is in sharp contrast to the
earlier reports of STM images of DNAs on
HOPG [21,221. These results indicated that the
AFM might be a useful tool in the study of
DNA-protein interactions, DNA packing, as well
as gene sequencing.
0304-3991/93/%06.00 0 1993 - Elsevier Science Publishers B.V. All rights reserved
15X J. Yrrng, Z. Shao / Effkt of probe force on the resolution of AFM of DNA
Fig. I. Typical AFM image of a 10 kbp plasmid DNA ob-
tained in air, using a commercially available Si,N, pyramid
tip with a spring constant of k = 0.06 N/m and a scanning
speed of 4.73 Hz. The probe force was about 2 nN. Along the
DNA molecules. the minimum measured width in many places
was between 3 and 4 nm, when scanned at higher magnifica-
tion (200-400 nm). The image was obtained under a humidity
of 35%.
So far, the limitation on the resolution achieved
on DNA molecules has been largely attributed to
the tip geometry [lo-121. The commercially avail-
able Si,N, pyramid tip cantilevers have an apical
angle of N 55 and a nominal tip radius of curva-
ture of - 30 nm [23,24]. By assuming a rigid
cylindrical chain for the DNA molecules, the tip
broadening effect was indicated [lO,ll]. It was
reported that, supertips having an apical angle
of less than lo and a reduced radius of curvature
at the tip yielded improved lateral resolution
[ 11,121 at otherwise similar conditions.
In this article, we present a comparative study
on the effect of the probe force and the environ-
ment on the lateral resolution of DNA molecules
by AFM using commercially available cantilevers.
We found that the lateral resolution is closely
related to the so-called adhesion force, and to a
much lesser degree to the probe geometry. This
result seems to indicate that some sort of contam-
ination on the tip, which should be directly re-
lated to the adhesion force, may be responsible
for the frequently observed degradation of reso-
lution. We will present a procedure for cleaning
AFM tips to reduce the adhesion force to as low
as 1 nN. This method can yield a resolution of
4-6 nm for most specimens in air, comparable to
that obtained by supertips in organic solvents
112,161. A practical approach using the adhesion
force measured on a distance-versus-force curve
as a diagnostic procedure was found quite suc-
cessful in the assessment of the quality of the tip,
as well as the specimen. All these results are
directly from the experimental findings, and a
complete theoretical analysis has not been devel-
oped.
2. Materials and methods
2.1. Specimen preparation
Our specimen preparation method is a simpli-
fied version of the Kleinschmidt method for elec-
tron microscopy [20]. Double-stranded plasmid
DNA (10 kbp), consisting of a cDNA encoding
the (Nat+ K+)-ATPase a-subunit and a mam-
malian expression factor, was used [9]. DNA solu-
tion (_ 2 pg/ml), containing 100 Fg/ml cy-
tochrome C, 1OmM Tris, 1mM EDTA, pH 8,
distilled and deionized water and 50% for-
mamide, was well mixed prior to spreading. A
glass rod of 3.3 mm diameter, cleaned by soaking
in chromic acid and rinsed thoroughly with dis-
tilled and deionized water, was positioned - 45
with respect to the horizontal level with one end
immersed in a teflon dent (made by l/2 drill)
filled with distilled and deionized water (- 0.5
ml>. A droplet of DNA solution (11 ~1) was
pipetted slowly onto the glass rod 2-3 cm above
the subphase water surface. The droplet should
flow down the rod freely, so that a protein film
was formed at the air-water interface. At certain
viewing angles, this protein-DNA film should be
observable to the naked eye. The glass rod was
slowly removed to minimize disturbance to the
protein film. A minute or so later a piece of
carbon-coated mica was used to pick up the
monolayer by touching the water surface. This
operation is performed in a humidity-controlled
environment with the humidity below 35%. The
J. Yang, 2. Shao / Effect of probe force on the resolurion of AFM of DNA 159
specimen was left to dry in air. For carbon coat-
ing, freshly cleaved mica was placed in a vacuum
evaporator (Denton Vacuum, DV502A). The
evaporation was carried out at low current (35-45
A) and long period (30-40 min) to ensure fine
carbon granules. The carbon-coated mica was
then placed in an oven baked overnight at 200C.
This procedure led the carbon film strongly bound
to the mica surface such that it cannot float off
the water surface when used to pick up the pro-
tein-DNA film. The baking is not mandatory, but
useful for more reproducible preparations.
Cleanness is critical in this method for high-qual-
ity specimens.
2.2. A FM imaging
The AFM images were obtained by a
NanoScope II AFM (Digital Instruments Inc.,
Santa Barbara, CA) with commercially available
cantilevers (k = 0.06 N/m, also from Digital In-
struments Inc.). For AFM in air, we found that
the probe force remained stable over a period
over 10 h for humidities below 40%. For imaging
in an organic solvent, such as isopropanol or
n-butanol (Sigma Chemicals, St Louis, MO), a
home-made fluid cell retrofitted to the Nano-
Scope II was used [6]. Different organic solvents
did not affect the quality of AFM imaging appre-
ciably, only that the evaporation rate is lower
with n-butanol. It is highly advisable to use a very
small scan area for initial approach (usually 5-10
nm). After the tip is engaged, the distance-
versus-force curve should be examined immedi-
ately to minimize the probe force applied. This
procedure will prevent specimen/tip damage by
the very large, initial probe force. Only if the
adhesion force is small, a larger area scan can be
started to search for DNA molecules. The force
curve should be constantly checked to correct for
drifting that can cause either accidental disen-
gagement or gradual increase of the probe force.
3. Experimental results
It was reported that, in the Kleinschmidt
method, the denatured cytochrome C molecules
Fig. 2. AFM image showing a defect on the cytochrome C
film, with the depth of - 1.3 nm. The defect was made by
scanning an area of 100x 100 nm2 at 78.13 Hz with a probe
force about 10 nN. The image was obtained after reducing the
probe force to - 3 nN using a scanning speed of 4.73 Hz, at
45 rotation to avoid any possible drag by the tip from the
build-up at the edge of the defect to influence the area with
the DNA. All above operation was carried out at a humidity
of 37%.
formed a thin layer at the substrate surface with
DNA adsorbed on top of it [251. Presumably the
DNA also binds to the denatured cytochrome C
molecules with details completely unknown [25].
This assertion of a denatured protein film on the
substrate is confirmed by AFM as shown in fig. 2,
where not only a DNA molecule is shown, but
also a 100 nm square surface defect, which was
made by using a large probe force that pushed
the denatured proteins to the edge of the scanned
area. This protein film, measured from fig. 2, is
about 1.3 nm thick in the dehydrated form, al-
though the degree of compression is not clear.
From the nature of this preparation method, we
can expect that the image quality, both the con-
trast and the resolution, should be quite sensitive
not only to the geometry of the tip, but also to a
range of other parameters, such as the probe
force, humidity etc. Due to the soft nature of
both the DNA and the protein film, it is clear
that only under a probe force that does not
exceed a certain limit, imaging can be successful.
IhO .I. Yang, Z. Shao / Effect uf prdw f&e on the resolution of AFM of DNA
.42
Fig. 3. Two force curves with corresponding AFM images are presented to show the effect of electrostatic interaction. In (a), the
electrostatic interaction became effective when the tip was about 1.8 pm away from the specimen surface. Since the magnitude of
this force was so large that it was out of the range of the instrument, only part of it is shown to demonstrate the long-range
characteristic of the electrostatic interaction. Two arrows indicate the tip approaching and leaving the specimen surface, which is to
the left of the display. The curvature of the curves is due to the electrostatic attraction. The set point marks the setting of the
cantilever during AFM imaging. The magnitude of this force ( - 17 nN) was estimated from the set point voltage change when the
tip was engaged. The same specimen, when grounded, showed absence of any long-range interaction. In (b), the force curve after
grounding is shown (notice the different scale). The force curve shows a straight line when the tip approaches the specimen until it
touches the surface (indicated by the arrow). When the tip withdraws, it disengages from the specimen at a position where the
restoring force of the cantilever overcomes the adhesion force. Such a hysteresis was well observed with specimens with a finite
adhesion force. In (b), the adhesion force is only - 1.2 nN. (c) AFM image with the force curve shown in (a); (d) AFM image with
the force curve in (b). Both (c) and (d) were obtained with the same tip on the same specimen. The humidity was 30%-35%.
Indeed, the control of the probe force is found
very important for AFM of DNA. But our study
also indicated that other factors arc equally im-
portant, if not more so. In the following, we
present experimental results which indicate what
factors must be carefully controlled in order to
obtain AFM images of DNA.
3. I. Electrostatic forces
Static charge often accumulates on the surface
of an insulator, with the amount sensitively de-
pendent on location, environment and surface
geometry. Therefore, it is virtually impossible to
even estimate its exact effect in a given experi-
ment. Mica, often used as the substrate in AFM
experiments, is known to carry some negative
surface charges after cleavage 1261. If a grounded
conductor is placed in front of such a charged
Fig. 4. Sketch to illustrate the grounding of the specimen. In
the NanoScope II AFM, the steel plate is grounded, so is the
metallic cantilever holder. The carbon paste will provide
grounding for the carbon film on the mica surface.
surfac ze, an attractive force will arise [27]. As we
founi %, with the carbon-coated mica, a long-range
force often occurs, as shown in fig. 3a. The de-
cline of the base line could be attributed to this
elect] :ostatic interaction [28], because the electro-
static charge on the surface can induce an oppo-
J. Yang, 2. Shao / Effect of probe force on the resohrtion of AFM of DNA 161
site charge in the gold coating on the cantilever
(grounded in the NanoScope II AFM) which
would result in an attractive force. This attractive
force can aIso be confirmed from the fact that, as
the cantilever was approaching the substrate sur-
face, the differential signal (A-B voltage output
19.94 nm/div
Fig. 5, AFM images of two DNA molecules at two different adhesion forces. Images (a) and (b) were obtained with a probe force of
- 1.8 nN and the adhesion force of - 1.2 nN shown in Cc); images Cd) and (e) show the same molecules under a probe force and
the adhesion force of - 17 nN. The change of lateral resolution by a factor of 3-5 on the same molecule is clearly shown. Scanning
speed for all 4 images was 4.73 Hz. Humidity was at 32%.
162 J . Yang, Z. Shao / Effect of probe force on the resolution of AFM of DNA
from the NanoScope II AFM) decreased to a very
large negative value before tip engagement. Fig.
3a shows that this force becomes effective when
the tip is 1.8 pm away from the surface. The
magnitude of this electrostatic force varies for
different preparations, but is almost always pre-
sent. With the electrostatic force shown in fig. 3a,
no stable images of DNA were obtained (fig. 3c),
possibly because the initial engagement has so
large a probe force that the tip punched through
the protein film right after engagement; it is very
likely to have some pieces from the specimen
attached to the tip or to have some damage on
the tip. Furthermore, with the force curve shown
in fig. 3a, the smallest probe force one could use
is no less than 15-20 nN (estimated from the
change of set point voltage during tip engage-
ment), otherwise the tip would disengage from
the specimen quite easily, due to the non-uni-
formity of the specimen. However, since the car-
bon film on the mica surface is conductive, any
surface charge can be totally shielded if the car-
bon film is electrically short-circuited to the
ground, as shown in fig. 4. By doing so, charges
on the carbon film are also removed, and charges
on the protein film will be balanced by the in-
duced charge in the carbon film (thus its effect is
very much reduced). Fig. 3b is the force curve
after grounding with fig. 3d as an example of the
effect of grounding for AFM imaging of DNA.
The long-range interaction is totally eliminated.
This procedure, as we found, is crucial for obtain-
ing any images of DNA with this kind of speci-
men. We should stress that the images in fig. 3
were obtained from the same specimen and with
the same cantilever under identical conditions,
with the only difference being the grounding. The
effect of grounding on image quality is quite
apparent when operated in air.
3.2. Adhesion force and its effect on resolution
For many substrates, such as mica, gold and
HOPG, there is always a quite large adhesion
force between the probe and the specimen sur-
face [29]. This force is often many orders of
magnitude larger than the van der Waals force,
which strongly pulls the probe towards the speci-
men surface. The origin of this force has been
attributed to the existence of a water layer at the
surface 1291. The meniscus formed between the
probe and the water surface would produce such
a force 130-321. However, for a protein film, such
as the specimens used here, it is not clear whether
the adhesion force is solely due to such a menis-
cus attraction.
We have found, through experiments, that the
success of AFM imaging of DNA is directly re-
lated to the magnitude of the adhesion force. Fig.
5 shows a pair of DNA molecules on the same
specimen and with the same tip under two differ-
ent adhesion forces. The force curves are also
shown below the DNA images for comparison.
The difference in the measured molecular width
is quite apparent: 4-5 nm averaged along the
DNA molecule for the small adhesion force and
about 15 nm for the larger one. To avoid confu-
sion, we define the lateral resolution as the half-
height full width along the direction perpendicu-
lar to the molecule throughout this article. When
the adhesion force was small, such as the one
shown in fig. 5c with a peak value of 1.2 nN, the
lateral resolution routinely achieved was 4-6 nm
when operated in air. With a large number of
specimens, we found there was a general trend
that a larger adhesion force was always related to
a lower resolution. A factor of 3-5 on the same
DNA molecules in measured lateral resolution
(as shown in fig. 5) was quite common. In order
to quantitate this effect, it would be ideal to use
the same tip on the same molecule while imaging
at various adhesion forces. Unfortunately, the
adhesion force cannot be controlled at will.
Sometimes, a reengagement of the cantilever
would cause an increase in the adhesion force,
with the increment mostly in the range between 7
and 30 nN. In addition, any given tip has a finite
usable life span. It is virtually impossible to use
the same tip for too long, because accidental
damage does occur during engagement or even
during imaging (particularly on poor specimens).
In fig. 6, we plotted the data of adhesion force
versus lateral resolution, obtained from a large
number of specimens and commercially available
cantilevers. Among the data shown, some were
from the same tip over several specimens at vari-
.I. Yang, Z. Shao / Effect of probe force on the resolution of AFM of DNA 163
1'
I 10
Adhesion Force (nN)
Fig. 6. Log-log plot of the adhesion force versus lateral
resolution. Each data point in this plot was obtained by
averaging along the DNA molecule of one specimen under
one adhesion force. The error bar is the standard deviation
which indicates the non-uniformity of cytochrome C binding.
For those data points with adhesion forces below 6 nN, the
lateral resolution was measured from small scan areas (200x
200 nm to 400~400 nm2). For those data points of larger
adhesion forces, the resolution was measured directly from
images of typically 1 x 1 Fm2. A least-squares fitting indicated
that the lateral resolution is proportional to (adhesion
force),48. See section 4 for a discussion of this result.
~_., .., . ,.. , ..~. ~ , ., ._~ (^.. ~ _,_.. ~ .,_.., ..,. , _.,
19.94 nm/div
ous adhesion forces. These data were accumu-
lated over a period of several months with some
specimens stored in a desiccator for up to 10
months. Each data point in fig. 6 was obtained
under one adhesion force averaged along the
DNA molecule. To avoid complications from
other factors, the humidity in the imaging cham-
ber was controlled to within 30%-40% for all
experiments included in fig. 6. Despite the fact
that the specimen preparation conditions were
not identical and that the tip geometry must vary
for individual cantilevers, the correlation between
the adhesion force and the lateral resolution is
clearly recognizable in fig. 6. The variation in the
lateral resolution for the same adhesion force
may be an indication of the variation in the tip
geometry and/or in the specimen preparation.
The latter is expected to give some noticeable
deviation for the lateral resolution, because there
is no control of the amount of cytochrome C
binding on the DNA which should definitely af-
fect the molecular width. To ensure the consis-
Fig. 7. Three images of the same DNA molecule under probe forces ot (a) 2, (b) 9 and tc) 12 nN, as shown by the force curves
below. For (b) and cc), dashed lines were drawn to indicate the relative levels of the probe forces, because they were out of the
range of the display. The horizontal dashed line for (b) and (c) were positioned according to a linear extrapolation of the set point
voltage calibration. The resolution remained approximately constant. But in (cl, the molecule appeared damaged in many places.
Scanning speed for these three images was 4.73 Hz. Humidity was 38%.
164 J. Yang, Z. Shao / Effect of probe force on the resolution of AFM of DNA
tency in these measurements and to reduce the
effect just mentioned above, only those speci-
mens that showed smooth and continuous
molecules were plotted in fig. 6. For adhesion
forces larger than 30 nN, DNA images were no
longer obtainable, even when the probe force was
set below the maximum value of the adhesion
force. A very practical conclusion from fig. 6 is
that the adhesion force may be used as a diagnos-
tic method to evaluate whether the imaging con-
ditions are proper for a given experiment. If the
peak value of the adhesion force is above 30 nN,
the best solution would be to change either the
specimen or the cantilever. Another conclusion is
that the commercially available cantilevers are
quite consistent in performance. Almost all the
cantilevers we have used can yield 4-6 nm resolu-
tion when properly cleaned as indicated by a
small adhesion force (see below for details). It is
interesting to note that, when the adhesion force
is below - 3 nN, the lateral resolution reached a
range of 4-6 nm, with many tips even capable of
a resolution of - 3 nm under optimal conditions.
Due to the binding of cytochrome C, it is not
clear whether this broadening is caused by the tip
size or the protein binding. But it is safe to
conclude that if the adhesion force is small, the
tip broadening is unexpectedly small for most
cantilevers, considering the radius of curvature of
most tips. Based on these results, the merit of a
supertip at this resolution scale is not apparent, if
one considers that, even in liquid, the supertips
only achieved a similar resolution [12,16].
3.3. Absolute probe force and adhesion force
The adhesion force, regardless of its origin, is
due to an interaction between the tip and the
specimen. Therefore it is intrinsic and depends
on the specimen preparation and the tip condi-
tion rather than the instrument. Logically, one
would assume that, even for a small adhesion
force, the resolution would deteriorate in a simi-
lar fashion as in fig. 6, if the probe force (the
absolute force) is raised to a similar level as the
peak value of the adhesion force. This can be
easily examined by changing the set point voltage
on the NanoScope II AFM. Surprisingly, the ex-
periment did not comply with this expectation.
Fig. 7 shows a series of 3 images with a probe
force of 2, 9 and 12 nN, respectively. Despite the
change in probe force by a factor of 6, the resolu-
tion remained roughly the same. However, at a
probe force of 12 nN, part of the DNA molecule
was damaged as indicated by the discontinuity
along the contour of the DNA molecule. Further
increase of the probe force to 15 nN resulted in a
sudden degradation in resolution, with an in-
crease of the adhesion force to 20 nN at the same
time. For all experiments conducted, it was found
consistently that the resolution is related to the
absolute probe force in an all-or-none fashion.
In other words, the resolution would remain al-
most the same, up to a threshold force at which
both the adhesion force and the molecular width
suddenly jumped to a larger value. These results
seem to indicate that the larger probe force ap-
plied to the specimen induced some kind of con-
tamination on the tip, presumably from fragments
of the specimen, which appeared as an increase
in the adhesion force. Our experiments consis-
tently showed that, for a larger adhesion force,
the specimen could normally withstand a much
larger probe force (up to 30 nN) compared with
the case of a very small adhesion force. These
observations are consistent with the results dis-
cussed in section 3.2. Apparently, the adhesion
force should be used as an indicator for the
condition of the tip during AFM imaging.
Rubber Glove Opening
Gas Outlet
Fig. 8. Illustration of the humidity control. Valves at dry
nitrogen inlet and gas outlet on the top of the humidity
chamber were used to control the speed of nitrogen gas influx,
therefore, to regulate the humidity. A rubber glove opening at
the top of the chamber was used to adjust AFM detector
position if necessary.
Fig. 9. 1
and (d)
J. Yang, Z. Shao / Effect of probe force on the resol uti on of AFM of DNA 16.5
19.94 nm/div 19.94 nm/div
I a ..z
.! ..> . . . .
:::: : ! ./ . ..i 1 (Arb Unit)
./ .i..
I .:::/ Q:!_ :/.. .: :_: ...I. / ,,y;:,
_i.
..i. ,....i .,.....: .,..i (.,,: . ... ./ :... :.., I i.
_ - ._ 7
,..../ _i. . . . .
-0 (Set Point)
.i
,:::: I 1;:. , 1:: ::::/ -j:. .::;
* :
. . . . i:: .j: ! f _;. .j: i ::I
; ..; ,. .!.,..... j ,:::
19.94 nm/div 19.94 nm/div
Four AFM images of the same DNA molecule with corresponding force curves, at humidities of (a) 37%, (b) 60%
90%. Neither the lateral resolution nor the adhesion force showed any appreciable change. Since the adhesion
not change with the humidity, it is not clear whether it is from the capillary force. Scanning speed was 4.73 Hz.
f
Cc) t 30%
orce did
166 J . Yung Z. Shao / Effect of probeforce on themolution of AFM of DNA
3.4. Effect of humidity
Although the effect on resolution due to the
adhesion force and absolute probe force was
studied under a controlled humidity, the effect of
humidity has been indicated to change both the
adhesion force [30-321 and the resolution of AFM
on DNA with different specimen preparatory
methods [11,33]. With the cytochrome C spread-
ing method, we carried out a series of experi-
ments in a humidity-controlled chamber where
the relative humidity can be adjusted continu-
ously (see fig. 8 for an illustration). To minimize
the effects due to other factors, such as tip con-
tamination, every effort was made to ensure that
the adhesion force was less than 3 nN before
humidity variation was started. To our surprise,
we found that within the experimental error, the
resolution of DNA molecules was unchanged over
a range from less than 5% to about 95% in
humidity. One example is shown in fig. 9 where
the images were taken at humidities of 37%,
60%, 80% and 90%, respectively, over a period of
- 7 h while the tip remained in contact with the
specimen surface. The corresponding force curves
are also shown at these conditions. At least for
this kind of specimen, the change in adhesion
force versus humidity is insignificant, which seems
to indicate that the so-called capillary force is
unimportant in this case. The reason might be the
protein film present at the surface which has
quite different properties from those materials
we are familiar with. We should also point out
that, for higher humidities (> 50%), long-time
disengagement of the tip often resulted in a much
increased adhesion force with much degraded
resolution when reengaged. However, as long as
the tip was engaged, high humidity did not show
significant degradation on image quality, even
over a long period of several hours.
3.5. Imaging in organic solvents
In organic solvents, like isopropanol and n-
butanol (also known as isopropyl alcohol and
n-butyl alcohol), both cytochrome C and DNA
are insoluble. Therefore, under such liquids DNA
molecules should be stable at the substrate-liquid
4.83 nmldiv
Fig. 10. AFM image of a DNA molecule taken in a fluid cell
under isopropanol, with the corresponding force curve. The
distance between two arrows in the force curve was about IO
nm, corresponding to a probe force of 0.6 nN for a cantilever
of k =0.06 N/m. Scanning speed was 4.73 Hz. Notice that
the adhesion force is absent.
interface. These solvents can also be used as
dehydrating agents. The role of dehydration in
AFM imaging is not clear at present. However,
there is one advantage for AFM of DNA in these
organic solvents: the capillary force, if any, is
entirely eliminated, so that the probe force can
be further decreased. For AFM of DNA in these
liquids, we found an interesting phenomenon:
usually within the first half hour or so after
engagement of the tip, no DNA molecules were
found and the probe force fluctuated a great
deal. 30-40 min later, the fluctuation gradually
damped down, and only then could the probe
force be set to sub-nN range. The adhesion force
was normally absent. Shown in fig. 10 are an
image of a DNA molecule in isopropanol and the
corresponding force curve. The resolution of
DNA molecules obtained in organic solvents is
quite similar to those obtained in air, i.c. 4-6 nm.
But, occasionally, on poorly prepared specimens
.I. Yang, Z. Shao / Effect of probe force on the resolution of AFM of DNA 167
Fig. 11. Close up of a DNA molecule taken under n-butanol.
Arrows indicate where a lateral resolution of - 3 nm was
measured.
that DNA appeared discontinuous in air, a reso-
lution around 3 nm was obtained (fig. 11, as
indicated between the arrows>. The clumps along
the DNA molecules may be the bound cy-
tochrome C molecules. Since the double helix
should have a diameter of - 2 nm (although 2.7
nm was reported by electron diffraction studies
[34]), the measured molecular width still shows
some broadening. It is not clear whether this
residue broadening is caused by the cytochrome
C molecules or the AFM tip itself. Should the
former be the primary contribution, the tip
broadening would be exceedingly small. It should
also be stressed that such a good resolution was
only obtainable with those specimens considered
rather poor for AFM imaging in air. Since the
amount of broadening is comparable to those
observed on native DNA molecules with super-
tips [12,16], whether a supertip will improve upon
this resolution with these specimens is not clear.
4. Discussion
Based on the experimental findings presented
so far, it is plausible to conclude that, at least for
the DNA specimens using the cytochrome C
spreading method, the contamination on the
probe tip is the single most important factor in
limiting the resolution achievable by AFM, rather
than the tip geometry it is often attributed to.
This conclusion may be understood by the follow-
ing: the adhesion force is related to the contact
area and the contamination near the tip would
increase this contact area. When the contact area
becomes exceedingly large, the resolution must
be degraded. If this is correct, we would have:
resolution a \/adhesion force. In fact, the data
presented in fig. 6 are quite consistent to this
notion with the power as 0.48 after a least-squares
fit. Furthermore, the observation that specimens
can only withstand a rather small probe force
(< 15 nN1 for a clean tip provided another
support for this conclusion, because for a contam-
inated tip (large contact area), the pressure would
be much smaller than a clean tip (small contact
area) for the same probe force, while the speci-
men damage should be directly related to the
pressure, rather than the force itself.
For a tip that showed a very large adhesion
force (contaminated), we found that scanning
the substrate (carbon-coated mica) can often lead
to a dramatic reduction of the adhesion force
which, in turn, provided a much improved resolu-
tion. Details of depositing a carbon film on mica
have already been described in section 2.1 and
the thickness of the carbon film is unimportant. It
was found that a scanning size of 10 pm or larger
was more effective, and the typical scan speed
was 5.79 Hz. Only when the adhesion force re-
mained small and stable (most often it did) dur-
ing subsequent scanning for at least 2 min, we
considered the tip as clean. For such a clean
tip, the adhesion force on the carbon film often
remained small (- 2 nN) for hours with only
small variations (< 10%). Most tips can be
cleaned two to three times. Interestingly, such
a procedure required the ambient humidity below
40%, and prolonged disengagement of the tip
could render the procedure ineffective. For higher
humidities (> 45%), such a procedure did not
work well. In these cases, the scanning of the
carbon film can still reduce the adhesion force.
But after the change of the specimen, the adhe-
sion force returned to the larger value. A DNA
specimen may also be used for this cleaning
J. Yang, Z. Shao / Effect of probe force on the resolution qf AFM of DNA
.:/. 2
, 1 (Arb Unit)
! j.. ;. :
i_/ -L
--_-.+- -- __/
I--L-lc- 0 (Set Point)
24.17 nm/div
,j,,,,i !.I
ail::- ~~111:1:~~~r-_ ~2
21.75 nm/div
3.02 nrhiv
Fig. 12. Four images of DNA molecules obtained by the same tip on the same specimen. (a) At an adhesion force of 27 nN. the
DNA molecule showed a resolution about 1X nm. (b) After cleaning the tip using the procedure described in the text, a typical
AFM image of a DNA molecule is shown, with an improved lateral resolution and a much reduced adhesion force ( - 1.4 nN). (c)
The same molecule as in (b) when the adhesion force was increased to 25 nN with a probe force of 22 nN. (d) Another cleaning
recovered the better resolution and the smaller adhesion force. Scanning speed for the above images was 4.73 Hz. Humidity was
30%.
.I. Yang, Z. Shao / Effect of probe force on the resolution of AFM of DNA 169
procedure, but the results were less consistent,
presumably due to the variation of the specimen
preparation and the soft nature of the cy-
tochrome C film. Fig. 12 shows an example. In
fig. 12a, we see an image obtained with a fairly
large adhesion force at a rather poor resolution.
In fig. 12b, a high-resolution image is shown
which was obtained on the same specimen with
the same tip after the cleaning process. The ad-
hesion force now is only 6% of that in fig. 12a,
while the resolution is improved by a factor of
3-5. During prolonged imaging, sometimes the
resolution became suddenly degraded, accompa-
nied with an increased adhesion force (fig. 12~).
In this case, another cleaning process can often
restore the image quality, as shown in fig. 12d
which was the result of the second cleaning.We
found that, following this procedure, the same tip
can be repeatedly used on several specimens.
The mechanism of the cleaning procedure
cannot be determined based on these experi-
ments. Although it is possible that the scanning
on carbon film caused some structural change to
produce some sort of a supertip, several argu-
ments would favor the interpretation that con-
tamination at the tip was removed by such a
procedure. First, that at higher humidities the
cleaning procedure did not work well and that
the cleaning was more effective with a short
specimen switching time indicated that the treated
tip had a finite life time standing in air and
shorter life time at higher humidities. This would
argue against a restructured supertip, which
should be insensitive to air exposure or humidity
change. It may be further argued that, at higher
humidities, water condensation on the tip should
be more significant that could backmigrate the
contamination pushed aside by the cleaning
procedure to the tip when suspended from the
specimen surface. Secondly, when imaging in or-
ganic solvents, the tips were not treated by the
cleaning procedure. Still most of them showed
high-resolution images of DNA, although it would
take some time after initial engagement (a few
minutes). This is consistent with the assumption
that contaminations on the tip, presumably some
organic materials, were dissolved away by the
solvent. Since the adhesion force does not change
with humidity appreciably, and high-resolution
images were also obtained in organic solvents, the
contamination is most likely due to hydrocarbons
abundant in air. If this is indeed the case, some
experimentation with different tip materials,
clean-room storage and operation, as well as tip
cleaning with other solvents or laser ablation,
may prove to be a fruitful approach. Despite the
above arguments, the possibility of structural
change due to such a cleaning procedure can-
not be entirely ruled out. A direct visualization of
the treated tip would be required before we can
fully understand the mechanism of such a proce-
dure
In conclusion, experimental results have shown
that the adhesion force is the most important
factor in obtaining high-resolution AFM images
on DNA. The commercially available cantilevers
are sufficient to provide resolutions up to 3 nm
for DNA and 1.8 nm for fully hydrated mem-
brane proteins [6]. Humidity control is much de-
sirable if the ambient humidity is above 40%.
Elimination of the electrostatic interaction is the
first crucial requirement for obtaining prelimi-
nary images on these specimens. As long as these
procedures are followed, reproducible high-reso-
lution DNA images can be obtained by AFM
routinely with minimal requirements on the envi-
ronment and the operator skill. Further improve-
ment on the resolution may require the use of the
so-called supertips. However, for the same probe
force, the pressure will be drastically increased.
whether these specimens can withstand such a
pressure to give even better resolution is un-
known. But, if the specimen strength eventually
becomes the limiting factor in resolution, other
techniques might be introduced, such as limited
chemical fixation and low-temperature AFM. The
most important conclusion is that, at room tem-
perature and nN force level, small broadening on
these DNA molecules can be achieved by AFM
with commercially available instruments.
Acknowledgements
We are grateful to Drs. A.P. Somlyo and A.V.
Somlyo for helpful discussions. We would also
170 J. Yang, Z. Shao / Effect of probe force on the resolution of AFM of DNA
like to thank Drs. H. Yu and K. Takeyasu for the
gift of DNA. This work is supported by grants
from the Whitaker Foundation, US Army Re-
search Office (DAAL03-92-G-0002), the National
Institutes of Health (POl-HL-48807), and the
Jeffress Memoral Trust (J-265).
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