Chapter 1

Artifacts and Practical Issues in Atomic Force Microscopy

Peter Eaton and Krystallenia Batziou

Abstract
As with any other microscopic technique, in atomic force microscopy (AFM), problems can arise. Some of
these happen due to improper use of the microscope by the operator, and some are due to particular
characteristics of the sample. Some occur depending on the type of instrument, or from probe damage.
Some of them are artifacts inherent in the technique. Knowledge of these issues is important for correct data
acquisition and interpretation, and in many cases, training in AFM is inadequate. In this chapter we show
examples of common artifacts in AFM and describe, where possible, how to overcome them. Other practical
issues important for best practice in AFM operation, such as noise reduction and data processing, are also
discussed.

Key words Artifacts, Distortions, Errors, Technique, Imaging, Force spectroscopy

1 Introduction

Without a doubt, atomic force microscopy is an extremely powerful

and useful technique. In the biological field, in particular, it has
proved its worth by enabling experiments impossible by any other
technique. However, like any other method, it has limitations and is
subject to artifacts that can give rise to errors in images and other
measurements. Such artifacts occur in all instrumental techniques.
For instance, in spectroscopy, peak broadening, shifting, and dis-
tortion can be caused by a wide range of factors [1]. Other types of
microscopy are also prone to exhibit various artifacts [2]. Examples
include chromatic aberrations, which occur in both optical and
electronic microscopies due to imperfect lenses [3]. This kind of
problem causes artifacts in the resulting image. The imperfections
in the lenses lead to corresponding imperfections in the probe (the
electronic or optical beam). Similarly with AFM, imperfect probes
lead to imperfect images, and this is the most common artifact seen
in AFM. In AFM, these probe artifacts are troublesome, because
since the probe usually comes into physical contact with the sample,
it can change during scanning, leading to problems in the image.

Nuno C. Santos and Filomena A. Carvalho (eds.), Atomic Force Microscopy: Methods and Protocols, Methods in Molecular Biology,
vol. 1886, https://doi.org/10.1007/978-1-4939-8894-5_1, © Springer Science+Business Media, LLC, part of Springer Nature 2019

3
4 Peter Eaton and Krystallenia Batziou

With other microscope types, the probe being a beam of radiation,

it tends not to be altered by the sample, and thus does not change
during imaging. On the other hand, probe problems in AFM are
easily remedied (by replacing the damaged or contaminated probe
with a new one), while for electronic microscopy it may mean a
lengthy realignment progress, or require an improved microscopy
objective for optical microscopy.
Probably the second-most common category of artifact in
AFM is that of scanner artifacts. An AFM scanner is an amazing
piece of engineering. It allows positioning and motion of the probe
relative to the sample with sub-nanometer accuracy and precision,
but the high effective magnification and sensitivity of the AFM
means small errors in positioning or translation in the scanner can
be glaringly obvious. AFM scanners are subject to a variety of
different artifacts, which depend somewhat on their design as dis-
cussed below. These can often, though not always, be avoided or
mitigated once the user knows what to look for.
A variety of other artifacts can also occur, some only detected
when making certain kinds of measurements, and some that can be
inadvertently introduced during imaging processing. In general,
most artifacts can be corrected or avoided by the careful user, but
the first step in this process is knowing how to recognize the
artifacts that can occur. Thus, it is highly recommended that as a
new user begins to generate data with the AFM, they familiarize
themselves with these possible sources of error in order to improve
their data. On the other hand, much of the material discussed in
this chapter has previously discussed in AFM training courses, and
following these discussions, many experienced AFM users have
found that they can reinterpret their data once they have been
acquainted with these effects. Artifact and problem recognitions
should be a fundamental part of instrument training.

2 Types of Artifacts

2.1 Probe Artifacts Probe artifacts come about when the shape of the probe is nonideal.
In AFM, as in many other methods, the data output always depends
on the nature of the probe. The image obtained in any microscope
is a convolution of the probe with the sample. In the case of AFM,
the ideal situation is to have the probe with a tip diameter smaller
than the highest resolution required. The probe should also have a
high aspect ratio, such that the probe can reach to the bottom of
any depression or pits in the sample surface, with the minimum of
contact between the sides of the probe and sides of sample features.
However, this is not always the case. Probe-sample convolution and
the effect of different probe sizes are illustrated in Fig. 1.
Sample feature dilation by the probe is an effect that always
occurs in AFM imaging, and can be minimized, but not totally
 Artifacts and Practical Issues in AFM 5

Fig. 1 Illustration of effects of probe dilation on the image formed in AFM. In each
case, the left images show the probe and sample feature, the right the image
formed. The first two images show the effect on a raised feature with a small
probe (a), and then with a large diameter tip (b). The lower two images show the
effects of imaging a depressed feature (a pit) with a small (c) and large diameter
tip (d). Raised features tend to be dilated by the probe, while maintaining
accurate height reproduction. On the other hand, depressed features will
become smaller, both in width and height

removed. As described above, for imaging of raised features—for

example, nanoparticles on a flat surface—dilation occurs in the x–y
axes, but not in z axis. While some effects can make z heights
inaccurate, in general, z measurements are highly accurate in
AFM imaging. Image dilation does not occur in the z axis for
these types of features, although measurements of pits or holes in
samples can be inaccurate. This fact, combined with the fact that
the AFM is much more precise in the z axis compared with the x–y
axes, means that accurate measurements of features are ideally made
in the z axis in AFM. Z axis uncertainty can be controlled by
instrumental noise levels, which are normally on the order of
0.1 nm. On the other hand, errors due to dilation in the x–y
6 Peter Eaton and Krystallenia Batziou

Fig. 2 Images showing the differences between imaging with a sharp (a) versus
a blunt (b) probe. Both images show the same sample (biaxially oriented
polypropylene film, BOPP), imaged with a sharp probe (a) and with a probe of
the same type that had been blunted by prolonged use (b)

plane can be very large (10 nm or more), and are difficult to

evaluate without probe shape characterization.
The images in Fig. 2 show an extreme example of sample
dilation. The figure shows height images of biaxially oriented poly-
propylene (BOPP), a polymeric film that is often used as a probe-
characterization sample [4]. The two images shown were measured
at exactly the same scale, but the height image in Fig. 2a shows
many more fibers, and they appear much finer than in Fig. 2b,
which was measured with a very blunt probe. In this case, the
features are dilated, and many features are not seen as they are
smaller than the resolution obtainable with this probe. A sample
such as this can be useful in diagnosis of imaging problems. An up-
to-date of suppliers of probe characterization samples can be found
on the internet [5]. It is highly recommended that AFM labs
should be equipped with a known probe-tip characterization sam-
ple of some kind, to enable diagnosis of probe-related issues. Sev-
eral other types of sample can be used for this purpose, including
gold colloids, porous aluminum, and thin films with many sharp
spikes [6, 7].
While dilation is almost unavoidable in AFM, ideally, the probe
will have an isotropic shape. That is, it should be approximately
cone-shaped with a spheroid tip. Under these conditions, dilation
will not lead to significant shape changes in most cases. However, if
the probe shape is not isotropic (non-spheroid at the end) any
feature imaged by that probe will lead to a feature in the image
with a distorted shape. All commercial probes, when purchased,
should have a more or less ideal shape. Either probe damage or
probe contaminations can lead to distorted images. Very com-
monly, some small part of the sample will adhere to a tip, and
 Artifacts and Practical Issues in AFM 7

Fig. 3 Examples of tip artifacts leading to repeating shapes in the height images. The samples were protein
nanoparticles (a and c) and a glass microscope slide coated with in proteins (b). All scale bars represent
500 nm

change its shape, leading to distortions in the images. Tip damage

can also occur due to catastrophic failure of the probe material due
to application of high forces [8]. This effect is different to simple tip
wear, which would normally reduce resolution without changes in
shape of the probe. Such tip wear would lead to greater dilation,
but no significant change in feature shape.
Artifacts that give rise to unusually shaped features in the
images can usually be recognized easily, once the user knows what
to expect. Several examples of images containing such tip artifacts
are illustrated in Fig. 3.
If an image such as those shown in Fig. 3 is obtained, it usually
indicates the probe is either broken or contaminated. If the probe is
contaminated or dirty, it might be possible to clean it, while broken
probes cannot be improved, and must be replaced with a new
probe. Cleaning contaminated probes is possible by several meth-
ods [9–12], although in practice the chance of a successful return to
pristine condition is quite low. The method that is simplest is to find
a soft sample, and perform a nanoindentation experiment, using
the AFM software, applying sufficient force to indent into the
sample. Ideally, this will result in the contaminants being deposited
on the soft sample leaving the probe clean. This has been carried
out with the fibrous polymer film BOPP [4], and also with gold
films [7]. However, there are several issues with this:
1. It is not usually possible to distinguish a broken probe from a
contaminated one.
2. Even with contaminated probes, cleaning might be impossible.
3. Even if the contaminants are removed, the probe will probably
already be somewhat blunted due to use.
8 Peter Eaton and Krystallenia Batziou

Fig. 4 Illustration of a double tip effect. Height image of DNA molecules, where
each molecule has an apparent “twin” caused by contamination of the probe.
The x–y scale of the image is 1.5
1.5 μm, and the image height is 2.0 nm

Overall, in most cases, the best thing to do is to swap to a new

probe. Nevertheless, there are some cases where attempting to
clean the probe is justified.
There are some special cases which are actually variations of this
kind of probe artifact.For example, it is possible to have a good
probe, which is accompanied by a “second probe,” which is mostly
likely made up of some contaminating particle. In this case, one can
get a “double tip” effect, where each image feature is accompanied
by a “twin feature” [13]. An example of a double tip effect is shown
in Fig. 4.
Another related effect can occur when the sample contains
large features of considerably higher aspect ratio than that of the
probe. In this case the user obtains an image containing one or
more features that look just like an AFM probe. While many AFM
probes are rounded at the tip, they are usually pyramidal in shape
beyond the very tip. Thus, the image will typically contain pyrami-
dal features when this occurs. Any image of pyramids occurring in
an AFM image must be analyzed with caution as they are often also
artifacts [14]. Sample features that give rise to this effect cannot be
imaged by the AFM using the probe in question. Use of a higher
aspect ratio probe might overcome the problem to a certain extent.
Diagnosis of this problem can benefit from knowledge of the shape
of the particular probe used. For example, many probes are square
or triangular pyramids, giving rise to images showing these shapes
in place of the true sample features.
Both Figs. 2 and 3 showed examples of dilation of sample
features by the probe, leading to artificially larger features in the
final image. But, as illustrated in Fig. 1, when imaging holes in a
sample, such pores will appear artificially smaller due to convolution
of the probe shape. An example image showing this is included in
Fig. 5.
 Artifacts and Practical Issues in AFM 9

Fig. 5 Illustration of the effect of imaging holes in a sample surface with a blunt tip. (a) Height image of a
calibration artifact which features an array of square holes imaged with a sharp probe. When the same area is
scanned with a blunt probe (b), the holes in the surface appear artificially smaller

Figure 5b illustrates how the holes in the sample surface

become smaller when imaged with a blunt or broken probe. In
this image, the probe was broken and we can also see repeating
triangular shapes in the sample. It’s worth noting that in this case,
the holes were sufficiently large that the probe reached all the way
to the bottom of the pits. This means that the pits were actually
measured to be the same depth in both images. However, for
smaller holes blunt probes will also reduce the depth of concave
features in the image. This is illustrated schematically in Fig. 1.

2.2 Scanner Artifacts In an AFM, the movement of the probe relative to the sample is
carried by a scanner, which translates a voltage produced by the
control electronics to a nano- or micro-metric movement. In nearly
all cases, these are based on piezoelectric crystals, which expand or
contract depending on the polarity of the voltage applied to them.
Since the coefficient of expansion of commonly used piezoelectric
crystals is commonly on the order of 0.01 nm per volt [15], it is
relatively simple with these devices to produce movements with the
nanometric precision required for an AFM.
An ideal AFM scanner would have a perfectly linear relation
between the voltage applied and the distance traveled in all three
axes, would not have any cross talk between movements in different
axes, would respond instantaneously to changes in voltage, and
would also cease movement immediately that the controlling volt-
age ceases changing.
In general, scanners do not produce such perfect movements,
but suffer from a number of imperfections, which give rise to
specific image artifacts which will be discussed in this section. Not
10 Peter Eaton and Krystallenia Batziou

all AFM scanners are the same, as many different designs are in use,
and some reduce or eliminate some of these artifacts.
Finally, apart from the image artifacts produced by piezoelectric
scanners, it is useful for the AFM operator to be aware that piezo-
electrics are quite sensitive materials. They are fragile, and easily
broken. They can also be depolarized by heat or exposure to water,
which will render them useless. Therefore AFM scanners should be
handled with care, and maintained in a dry, room temperature
environment at all times.

2.2.1 Nonlinearity The first major scanner-related artifact is nonlinearity in the x–y
plane. In general, the response of piezoelectrics to voltage is non-
linear. While there are a number of ways to ameliorate, or reduce
this effect, which will be discussed below, if uncorrected this can
introduce severe distortions in the image. Figure 6 illustrates the
effects of this nonlinearity on an image of an AFM calibration grid,
consisting of square, regularly spaced holes in a flat silicon surface.
The two images in Fig. 6 show the same area of the same sample,
scanned using the same instrument and the same AFM probe. The
height image in Fig. 6a was measured using the raw output from
the scanners to linear voltage ramps. It can be seen that in this case,
the response in the X-axis was extremely nonlinear, while linearity
in the y axis was somewhat better but not perfect. The image shown
in Fig. 6b was linearized using position sensors.
There are a number of ways to remedy or improve the linearity
in AFM scanners:

Fig. 6 Example of distortion in the x–y plane of an AFM image due to nonlinearity in the scanner response. The
two images show the same area of the sample. (a) Height image recorded with hardware linearization
disabled, and (b) height image recorded with hardware linearization (displacement sensors) enabled. Both
images are approximately 74
74 μm in x and y and 235 nm in z (height)
 Artifacts and Practical Issues in AFM 11

1. Some AFM scanner designs are inherently more linear than

others [15]. For example, flexure scanners are commonly
used in some instruments to improve x–y linearity. These scan-
ners do employ piezoelectrics for movement, but the flexure
design reduces non linearity. There are actually quite a wide
range of AFM scanner designs, each of which will tend to give
different characteristic nonlinearity in the scanner movement,
unless corrected.
2. It is possible to employ software-based correction of nonline-
arity. The success of this approach will depend on the sophisti-
cation of the algorithms used to correct for linearity, and also
on the complexity of the distortions inherent in the particular
scanner design. A downside of this approach is that it makes
scanner calibration more complex, and less likely to give highly
accurate results. Nevertheless, it can give good results, and
many popular commercial AFM systems use this approach,
especially older models.
3. The third method is to use what is known as a closed-loop
scanner design. In addition to piezoelectrics to enable the
motion of the probe relative to the sample, displacement sen-
sors are included in the scanner, to measure how far the scanner
actually moves. This information is fed-back to the scanner, to
ensure it moves the exact distance required. This approach is
very effective, and is probably the best method overall. The
image shown in Fig. 6b was measured with displacement sen-
sors and a closed feedback loop. In Fig. 6a, this feedback loop
was disabled, and the resulting distortion can be seen in the
image. Displacement sensors add some complexity to the scan-
ner and electronics design, and as such can be somewhat more
expensive systems. In addition, in some cases, the displacement
sensor’s sensitivity is not fine enough for optimal performance
of the AFM, so open-loop operation (i.e., with feedback from
the sensors turned off) leads to lower noise levels in the images.
In these cases, it is advisable to turn off closed-loop operation
when high resolution or high sensitivity is required. However,
some modern systems feature displacement sensors with very
low noise such that this is unnecessary. If the user suspects that
XY nonlinearity is causing a distortion in the image, and does
not have the option of software or hardware linearization in
their system, the best option is to image a square grid, and
assess the nonlinearity in each port of the image, to determine
the best part to use for scanning. For example, in Fig. 6, it is
clear that the image in Fig. 6b is more linear than the image in
Fig. 6a. Analysis of such an image can also help to determine
the error level in X–Y measurements. Some software packages
can even use this data to perform post-acquisition correction of
images containing X–Y distortion.
12 Peter Eaton and Krystallenia Batziou

2.2.2 Creep Creep is the phenomenon that occurs in piezoelectrics whereby

after a certain movement is made, the piezoelectric tends to con-
tinue to move, even after the voltage stops changing. The practical
effect of this is distortion at the beginning of an image. For exam-
ple, if the image is measured top-to bottom (in the slow scan axis),
then the top few lines will suffer a distortion (Fig. 7b).
It is very simple to avoid this artifact, even in open-loop opera-
tion, however. The simplest method is to ensure the piezo is placed
in the position at which imaging will begin, and wait for the system
to stabilize (effectively reaching point “S” in the plot on the left of
Fig. 7a. This might take 30 s. An alternative is to begin the imaging
in “2D” mode, i.e., measuring profiles continuously, with the
profile being at the start of the desired scan area. Simply watch
the profile over time—if creep is occurring, the features being
scanned will move. After a while, they should stop moving, and it
is a good time to begin the full “3D” scan. Note that this might not
work well in the case that thermal drift is occurring (see Subheading
2.5—Sample drift for this effect).
In systems that continuously scan top to bottom then bottom
to top (as is the case in several commercial systems), this effect will
be minimized by the second scan. Note, that as this effect only
affects part of the image, the data can be corrected post-acquisition
by simply cropping out the affected area (assuming it can be iden-
tified), although some data will be lost in this process.

Fig. 7 The causes and effects of creep. (a) Schematic illustration what happens when a piezoelectric
transducer has a voltage change applied to it—the movement of the piezoelectric tends to continue even
after the voltage stops changing. At the point marked “S,” the creep has stopped, and the system is stabilized,
and suitable to begin measuring an undistorted image. In (b), the result of this effect on atomic force
microscopy images is illustrated. The distortion at the start of the height image (the distortion is seen at the
top, in this figure) is due to creep. The image (b) is approximately 9.5
9.5 μm in x and y and 380 nm in
z (height).This effect should not occur if closed-loop operation is used
 Artifacts and Practical Issues in AFM 13

2.2.3 Hysteresis (Edge Another effect caused by imperfections in piezo responses is hyster-
Overshoot) esis. Hysteresis in any material is the tendency to fail to return
perfectly to its original shape after extension or compression. In
the piezo actuator in an AFM, this means that in the x–y axis,
forward and back images may be slightly misaligned, although
this is not an important effect for most measurements. Hysteresis
in the z piezo is more important, since it can lead to inaccuracies in
some height measurements, and also in force-distance curve mea-
surements. Examples of the effects of this phenomenon are shown
in Fig. 8.
If the scanner is prone to this effect, it is actually hard to avoid.
The extent of its effects on the image can be lessened somewhat by
scanning more slowly, but will not disappear. The best way to avoid
it, if possible, is to use z sensor height data instead of raw height
data. This is only available if the instrument in use has a displace-
ment sensor on the z piezo. Use of the z sensor data will also
improve the accuracy of force-distance curves. In both cases, it’s
worth bearing in mind that if the z sensor has a considerable noise
level, using this data instead of the standard z height data may result
in better accuracy but lower precision.

Fig. 8 Effects of hysteresis in piezo actuators in AFM. (a) The image is from a height measurement over
square-profile posts that shows where errors can occur when passing over steep features. (b) An example of
an uncorrected deflection-distance curve (from which force-distance curves are calculated. Close to the
turnaround point at the right (arrowed), the forward and back traces do not match due to piezo hysteresis
14 Peter Eaton and Krystallenia Batziou

2.2.4 Scanner Calibration All AFM systems require calibration of the scanner movement in
order to deliver quantitative results. Commercial systems should be
delivered with an accurate calibration, and a certification indicating
the error in the calibration measurement. Nevertheless, piezoelec-
tric materials age over time, which means that, the exact number of
nanometers they move per applied volt changes. Typically, this
change is faster at the beginning of a scanner’s lifetime, and stabi-
lizes over time. It is highly recommended that the scanner is cali-
brated against a trusted calibration artifact 6 months after
installation, and every year thereafter. Calibration procedures have
been extensively described elsewhere [16].

2.3 Noise Vibrations in the AFM system inevitably give rise to noise in the
data produced. This is common in all high-resolution microscopes.
2.3.1 Acoustic
In AFM, compared to electronic microscopes, the system can be
and Mechanical Noise
more troublesome for two reasons. Firstly, the AFM is often a small
system, so it’s more prone to mechanical vibration. Secondly, in
AFM, the probe physically touches the sample, forming a mechani-
cal loop, which transmits all vibrations in the system into the probe-
sample interface. Typically, AFM installations include some form of
vibration isolation, which reduces these effects to an acceptable
level. Acoustic isolation might be included as well. Nevertheless,
these measures only reduce the level of noise. Figure 9 shows image
recorded in an AFM system located in a fully working vibration, and
acoustic enclosure. In Fig. 9, parts A and B are images recorded in
quiet conditions. Figure 9 (parts c and d) are similar images
recorded with people in the room talking and performing various
noisy activities. These produce significant transient noise in the
image.
In Fig. 9, the noise is clearly visible in the lower right hand error
image (Fig. 9d), and somewhat harder to perceive in the
corresponding height image, at the top right (Fig. 9c). But height
images do contain marked vibrations. These are illustrated in the
height profiles in Fig. 10, which were extracted from the images a
and c from Fig. 9.
In order to avoid the problems shown in Fig. 9, it is important,
firstly, to ensure the AFM instrument is shielded toward both
acoustic and vibrational influences. There are many commercial
vibration solutions available, both active and passive. Passive solu-
tions generally work by connecting the AFM instrument to a large
mass on the end of a soft spring. In this way, high frequency
vibrations are absorbed by the mass-spring system. Active systems,
on the other hand, use accelerometers to measure vibrations com-
ing into the system, and actively compensate for these using actua-
tors. Either kind of system can work extremely well to reduce (but
not to eliminate) vibrations entering the AFM system. Thus it’s
important to install the AFM in a low-noise environment, and avoid
the kind of transient impulses illustrated in Fig. 9. Acoustic
 Artifacts and Practical Issues in AFM 15

Fig. 9 Examples of transient noise effects in AFM images. All images show the same area of an HOPG sample.
(a, b) show height and amplitude images, respectively, of the samples taken under quiet conditions, i.e., with
the AFM enclosed in a vibration isolation cabinet, and no acoustic or vibration sources in the room. (c, d) show
the equivalent images of the same area with talking and noisy activities occurring in the room. The lines
marked P1 and P2 refer to profiles through these images shown in Fig. 10

Fig. 10 Height profiles indicating interference in height profiles from acoustic and vibrational noise. The graphs
show two line profiles (P1, P2) from Fig. 9a, c. In both graphs, the blue solid line shows the height profile
measured during noisy conditions (from Fig. 9c), while the red dashed line shows the same line recorded
under quiet conditions (from Fig. 9a)
16 Peter Eaton and Krystallenia Batziou

shielding can be sufficient to effectively reduce low-amplitude

noises, such as talking, and can be constructed fairly simply, typi-
cally consisting of a wooden box with foam on the inside. Measure-
ment of the noise floor of the AFM is typically used to assess the
success of vibration and acoustic noise reduction. The method to
measure noise floor has been discussed elsewhere [11]. AFM
instruments benefit from being installed on lower floors, away
from foot traffic and heavy machinery to reduce noise.

2.3.2 Electronic Noise It is possible for the AFM to pick up electronic noise from the
surrounding environment (e.g., radio-frequency interference), or
from an internal fault in the AFM to give rise to electronic noise in
the image. External noise sources can be diagnosed by switching off
possibly interfering devices. Fluorescent lights are a common cul-
prit in this regard, either malfunctioning, or not. Internal problems
in the AFM may be harder to deal with, but it’s important that the
user check for loose wires if such a problem is suspected. Very
commonly such problems are simple to diagnose, since they are
either completely constant, or switch on and off suddenly, unlike
other sources of noise, and have fixed frequencies. Commonly, this
will be the frequency of the mains AC source (i.e., either 50 or
60 Hz), which can be measured in the AFM image (Fig. 11).

2.3.3 Bit Noise “Bit noise” is an effect due to the limited bit resolution in the
analogue to digital converters (ADCs) which are a vital part of the
AFM. Usually this is only seen in z height images, when looking at
extremely flat samples, or measuring very small images. It comes
about because the AFM electronics usually record the height data
as 16-bit numbers. With 16 bits, the number of discrete height
values that can be registered is 216, i.e., 65,536. While this is a very
large number, and is sufficient for most cases, since the AFM is
extremely sensitive in z, it can be a limitation. If we take the case of
a very large scanner, with 10 μm height range, we can determine
that the smallest value that can be distinguished is 10,000/
65,536, i.e., 0.15 nm. This is a pretty small measurement, espe-
cially in a range of 10 μm; however, most AFMs will have a noise
floor several times smaller than this value. Thus for high-resolu-
tion imaging, bit noise can be a significant factor in image quality.
Fortunately, it is a limitation which is generally easy to overcome.
One solution is to use a smaller scanner. AFM instruments are
often purchased with multiple scanners for imaging samples with
difference size features. This does work, but it’s an expensive
solution to a simple problem with a simple solution. With most
systems, it’s possible to artificially limit the range of the scanner
used when imaging under high-resolution/low-noise conditions.
This has the effect of greatly reducing the bit noise. For example,
in the case above, the same scanner might be used with the
effective range of only 1 μm. Thus, the sampling would occur at
 Artifacts and Practical Issues in AFM 17

Fig. 11 Example of electronic noise in an AFM image. In this case, the noise that
can be seen at the bottom of the image was highly reproducible. It always
occurred in the lower part of the image, irrespective of the sample scanned, or
the conditions used. The cause is likely a loose connection in the scanner.
Thanks to Gleb Turlakov for permission to use the image

0.015 nm, or almost a tenth of an angstrom, practically small

enough for any application. Typically such a change can be made
in the AFM control software.

2.4 Image Image processing in AFM is very important. In fact, the directly
Processing Issues digital nature of AFM data makes image processing inherently easy.
Standard topographical AFM images are in fact a matrix of height
values. This makes the extraction of quantitative date from the
image relatively easy, although some care always needs to be taken.
Considering AFM height images, the data is, in general, stored
as 16 bit values. Therefore, there are 65,536 possible height values.
In other words, if we attempted to represent all this data on
computer monitor as a grayscale image, we would need to show
more than 65,000 shades of gray. There are currently no computer
monitors capable of this bit-depth. Even if they were, it would not
be possible for us to perceive this level of detail, since our eyes are
capable of distinguishing between 50 and 100 shades of gray
[17]. The same goes for printed images. An example of this is
shown in the Fig. 12. In Fig. 12a is shown a height image, with
the height illustrated using a grayscale, the most common form of
display used for AFM images. On Fig. 12b is a 3D-rendered and
light-shaded version of the exact same image. It is possible to see
18 Peter Eaton and Krystallenia Batziou

a 388 nm
b
350

300

250

200 0.39 µm
0.00 µm
150

100 y:
2.4 µm
µm 2.4
50 x:
500 nm
0

Fig. 12 Illustration of the difficulty of visualizing details in color-coded AFM height images. The two images
derive from the same data set. The conventional way to show AFM height data is in (a). This “color mapping”
representation is useful because the colors can be directly related to height values. However, many height
details are hidden since only a few shades of gray can be represented. The light-shaded rendering in (b)
shows many of these hidden details

many details in this shaded image which are “hidden” in the gray-
scale height image.
It is important that the user be aware that it is not always
possible to see all the details available in an AFM image at first
sight. Thus it is imperative that any modifications made to the data
via processing steps, such as histogram adjustment, shading, level-
ing, and filtering, be made non-destructively. It is critically impor-
tant that the original data file as saved by the instrument software is
not altered in anyway, as this can destroy data forever.
In this section of the chapter, some issues involving data pro-
cessing are discussed, with particular attention paid to processing
steps which could result in artifacts or image degradation.

2.4.1 Leveling Leveling is the most important step in AFM image processing,
which is applied to nearly all images [18]. Depending on the
image, it can be either extremely simple or quite difficult to get
right. One of the most common artifacts associated with leveling is
illustrated in Fig. 13. In this case, the effect is introduced by using a
polynomial leveling routine, which attempts to level the back-
ground, without taking account of features that do not form part
of the background. In Fig. 13a, an image of nanoparticles
distributed on a flat surface has been leveled with a horizontal
second-order polynomial fit. This results in the dark streaks or
shadows around the particles. If an adequate leveling routine is
used [18, 19], the image on the right can result.
The image in Fig. 13a does show correctly the positions of the
nanoparticles, but misrepresents the shape of the background.
However, there is a more serious implication of using inadequate
 Artifacts and Practical Issues in AFM 19

Fig. 13 Example of artifacts introduced into an image by a poor choice of leveling parameters. (a) A height
image of nanoparticles which has been leveled by a horizontal second-order polynomial fitting routine. As a
consequence, the particles are accompanied by dark streaks, since the routine fits the particles as if they were
a part of the background. (b) A correctly leveled version of the same image

leveling. Height measurements of nanoparticles made on incor-

rectly leveled images will be inaccurate. An illustration of this is
shown in Fig. 14. The profiles shown in Fig. 14 were made passing
over the center of the same nanoparticle from the images shown in
Fig. 13.
The data in Fig. 14 illustrate that the height of features in AFM
images can depend on performing leveling procedures properly.
There are a large number of algorithms that can level AFM images
without introducing such artifacts. For example, in the case of
Fig. 13, the nanoparticles could be masked out, and only the
unmasked data (i.e., the background) used for leveling. This results
in an image similar to that shown in Fig. 13b. For a more complete
overview of leveling procedures in AFM see refs. 18, 20.

2.4.2 Filtering Most AFM processing software allows the user to filter the images,
for a variety of reasons. One very common reason why users could
use filters on their images is to remove noise present in the data.
While this may be useful for display purposes, it’s important that
the user be aware that all filtering alters the data. Thus, any analysis
to be performed on the AFM data should be made using un-filtered
data only. As explained above, in many cases, subtleties in AFM
images are hard to discern by eye. Thus, it can be that an image
looks similar after filtering (albeit with lower noise), but on close
examination, the shape of features in the image may be considerably
altered by the filtering process. An example of the kind of alteration
that can occur upon filtering is illustrated in Fig. 15.
In Fig. 15, it’s possible to observe that a low-pass filter enables
smoothing, and reduction of noise, but can also change features
20 Peter Eaton and Krystallenia Batziou

bad levelling good levelling

150 150

100 100
vertical
nm

nm
101 nm 124 nm
50 50

0 0
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.0 0.1 0.2 0.3 0.4 0.5 0.6
150 150

100
100
horizontal
nm

118 nm
50
nm
127 nm
50
0

0
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.0 0.1 0.2 0.3 0.4 0.5 0.6

Fig. 14 Examples of vertical and horizontal cross-section profiles over the same nanoparticle from two
differently processed images coming from the same AFM height image (Fig. 13). On the left profiles from the
image shown in Fig. 13a; on the right are shown images from Fig. 13b. It can be seen that the way leveling is
performed can affect the measured height of features

significantly in the data. Most importantly, height data in the image

is changed. Particles that measured over 10 nm in the original data
(Fig. 15a, c) measured less than 8 nm in the filtered data (Fig. 15b,
d). Filtered data should never be used for further analysis.

2.5 Other Issues There are many sample preparation issues that can affect the results
obtained in AFM imaging. Proper sample preparation is vital for
2.5.1 Sample Adhesion
AFM measurements, and typically problems in sample preparation
cannot be overcome when imaging. Thus this first step in an AFM
experiment can be considered one of the most important. One issue
that can affect AFM imaging is cleanliness of the sample. AFM is a
surface characterization technique. Thus, a very thin (nanometric)
contamination layer could completely eliminate the sample features
which the user desires to scan. Dirty or contaminated samples can
also lead to probe contamination, which can also prevent imaging
as described above. Another issue is that the features to be imaged
should not move about under the forces from the probe. Most
commonly if this happens, horizontal streaks will appear in the
images, and the sample features will not be imaged at all. On
some occasions, however, the sample features will move only
slightly, leaving tracks across the image. This is illustrated with the
height image of nanoparticles in Fig. 16a.
 Artifacts and Practical Issues in AFM 21

Fig. 15 Example of the effects of filtering. The original height image (a) and a cross-section profile (c) through
several nanoparticles. A low-pass (smoothing) filter was applied, giving the result on (b). Shape and height
changes can be seen on (b and d, respectively) indicating that this data is not suitable for further analysis

Fig. 16 Height images illustrating the effects of marginal sample-substrate adhesion. The images show a
sample of nanoparticles on a surface to which they adhere poorly. (a) The first image obtained which shows
“drag marks,” caused by the samples being nudged down the image (the slow scan direction was down). This
results in image artifacts and underestimation of the number of particles present. (b) The same area imaged a
second time with higher PID gains, and lower force applied, which does not show dragging of the
nanoparticles
22 Peter Eaton and Krystallenia Batziou

The best way to overcome this effect is to ensure that the

sample features to be imaged are well-adhered to the sample sub-
strate. A number of different substrates are available for AFM
imaging, and changing from one substrate to another may alleviate
the problem [21–23].
If this effect is noticed, it can sometimes be overcome by
optimizing imaging parameters. In the example above, the applied
force (via the setpoint) as well as the feedback gains (PID) values)
were optimized to enable imaging the particles, without moving
them about. In fact, observing the height image in Fig. 16b, we can
see that imaging in this case was not perfect, the particles, being
slightly elongated in the fast scan direction (i.e., they look a little
like triangles instead of circles). This illustrates that if sample prep-
aration is not perfect, ideal imaging might be impossible.

2.5.2 Laser Interference
This kind of artifact is caused by reflections of the laser originating
from the sample surface. As illustrated in Fig. 17a, the laser light
that follows this path will tend to interfere with the laser that is
properly reflected from the back of the cantilever. In principle, if the
optical setup of the AFM was perfect, there would of course be very
little signal reflecting from the sample reaching the photodetector.
In particular, this interference is reduced because the cantilever is
usually at an angle compared to the sample surface (in most systems
this angle is around 10–12 ). This difference in angle can be
enough to direct laser light from the sample away from the photo-
detector. Moreover, most of the laser light should be tightly
focussed on the cantilever, with very little light “spilled” over the
edges of the lever. However in the real world, there are several
reasons why this can in fact occur. Firstly, many lasers in AFM

Fig. 17 (a) The principle behind laser interference in AFM. The single incoming laser beam can reflect from the
sample as well as the cantilever, creating interference at the photodetector. (b) The effect of such interference
on imaging. Vertical “waves” appear in the height image while scanning micron-sized areas. Typically the
wavelength of the artifacts is the same as that used in the laser. The sample is a silicon surface with deposited
nanoparticles. The x–y scale is 15 μm and the z scale is approximately 10 nm
 Artifacts and Practical Issues in AFM 23

systems are not as well focussed as y should be. Secondly, the user
might accidentally set the focus position of the laser on the edge of
the probe, which will allow some light to spill on to the sample. In
addition, many AFM cantilevers are actually semitransparent to the
laser light, allowing a small amount of it to pass straight through.
This particular problem is reduced if the cantilevers are coated.
Many levers are available coated with a gold film on the backside
for increased reflectivity.
The effect of this issue is illustrated in Fig. 17b. Typically
vertical waves appear in the image, with a wavelength dependent
on the laser used. The height scale of the image in Fig. 17b is about
10 nm, so it can be seen that this effect is quite small. Thus it does
not significantly affect large-scale images. However, for images with
low z scales, it can be troublesome. The simplest solution is to
realign the laser, ensuring as little overspill as possible. It is worth
noting that this particular effect also affects force-distance curves.
These are measured during a vertical movement of the probe
relative to the surface. Considering the diagram in Fig. 17a, we
can see that vertical movement is also likely to result in changes in
interferences. In fact, this effect is seen more often in force curves
than in images. An illustration of this is shown in Fig. 18.
Just as with the effect on imaging, the best way to avoid this
issue is to carefully realign the laser, or avoid using a reflective
substrate. This artifact can make further analysis of the force-
curve more complicated, but many force-curve analysis software

Fig. 18 Example of the effect of laser interference on a force-distance curve. The

wavy baseline is caused by interference between the two possible laser paths
24 Peter Eaton and Krystallenia Batziou

packages include procedures to overcome the disadvantages of this

effect, typically by fitting the baseline to a straight line. Many AFM
instruments are fitted with low coherence of modulated laser light
sources which effectively reduce all laser interference effects.

2.5.3 Feedback Incorrectly set feedback gain parameters can cause considerable
Parameters problems in AFM images. These parameters are sometimes
known as PID, or proportional, integral, derivative values. In real-
ity, tuning of the PID parameters is a basic requirement of AFM
operation, so an experienced AFM user should be accustomed to
adjusting and refining these numbers. However, many factors can
alter the specific values required, including sample topography,
scanning speed, scan size, etc. Therefore it is rather important
that the user is used to recognizing when these parameters need
adjusting. Figure 19 shows different images of the same region of a

Fig. 19 Examples of the effect of using incorrect feedback gains. Panels (a) and (b) are height and error signal
images, respectively, of a sample obtained using adequate feedback gains. Panels (c) and (d) show
corresponding images of the same area, obtained with the same settings, except for the feedback gains
(PID values), which were set too low. It is noticeable that the topography is not followed properly by the probe
in this case. All images are 4
4 μm in x and y
 Artifacts and Practical Issues in AFM 25

particle sample, which were measured with correctly (a, b) and

incorrectly set (c, d) PID values, respectively.
These images show some of the effects of having incorrectly set
PID values. There are several signs in images c and d from Fig. 19
that indicate than these gain values were set too low. For example,
in image c, the particles seem to all have “tails” pointing toward the
right. This image was collected in the left to right direction, which
means that the probe did not follow the downside of the particles
well. In the places where these tails appear, in the error image, we
see featureless dark “shadows.” These are an indication that in these
places, the probe is not touching the surface. Large featureless areas
in the error image are a sign that the probe is not following the
surface well. This occurs more in intermittent contact modes (such
as that used here), than in contact mode. Finally, comparing the
lower with the upper error images (i.e., Fig. 19b, d, respectively),
we see a much larger signal in the error image d. Larger error signals
indicate poorer feedback. To fix these problems would require
simply increasing the PID controller gains.

2.5.4 Uncontrolled Z This is an effect which is unique to the intermittent-contact mode

Oscillation of AFM (commonly known as tapping mode). The effect is seen as
large oscillations in the image, usually restricted to certain regions
of topography to certain materials. If these large oscillations are
seen throughout the image, the cause is usually another source of
noise. This effect is thought to be due to extremely strong probe-
sample interactions, which overcome the feedback system’s
attempts to smoothly follow topography. A common source of
such an effect is the presence of water or other liquids when
scanning in ambient conditions, either on the surface or on the
probe itself [24]. An example image illustrating this effect is shown
in Fig. 20.
This artifact can be a difficult problem to overcome. If it’s
suspected that localized water is to blame, the sample should be
dried further. On the other hand, the water may also be present on
the probe. Often this effect can be overcome by increasing the
energy in the probe, and decreasing the probe-sample interaction
force. This means using a larger free amplitude, and a smaller
amplitude reduction setpoint [24].

2.5.5 Sample Drift Sample drift refers to uncontrolled movement of the sample while it
is being scanned. As the term “drift” implies, this tends to be a slow
movement. For large images, this effect often causes very few
problems. However, slow movements tend to have serious implica-
tions when scanning at high magnifications—i.e., small scale
images. Sample drift is an important problem in all kinds of high-
resolution microscopy. For example, in electron microscopy, a lim-
iting factor when obtaining high-resolution imaging tends to be
26 Peter Eaton and Krystallenia Batziou

Fig. 20 Example of uncontrolled z oscillations. The effect is seen around the base of the bacteria in these
images, probably due to incomplete sample drying. The effect is seen most easily in amplitude or phase
images (amplitude image, b), but also occurs in height images (a). The images are 10
10 μm in x and y, and
the height image is approximately 1.3 μm in z

that the micrograph must be obtained very quickly, reducing image

quality [25]. Otherwise, if a slow acquisition is made, the image will
tend to be distorted, by being stretched in one direction. AFM
suffers from the same problem, especially because image acquisition
in AFM tends to be slow. Fortunately, in general, this problem
tends to impact imaging significantly only when scanning quite
small areas—for example, scan sizes of 500 nm to 1 μm and
below. In general, for these scan sizes it is possible to scan consid-
erably faster than usual since there tends to be only small topo-
graphic contrast when scanning such small areas. This leads to a
general rule of AFM imaging—small areas should be scanned at a
high scan rate, while large areas generally need a lower rate, due to
large topographic contrast, and high probe velocities. Sample drift
is generally thermally induced. That is, the sample is generally
expanding or contracting due to some thermal gradient. This
leads to several important considerations:
1. Thermal drift can be reduced by simply waiting.
If high resolution is required, and thermal drift is significant, it
can be advantageous to setup the experiment and wait for
several hours before beginning, to allow thermal equilibration
to occur.
2. Some samples will drift faster than others.
Metallic and metal-coated samples tend to drift more quickly
than others.
3. Removing sources of heat can help.
Principally, light sources in AFM instruments can contribute
significant heat.
 Artifacts and Practical Issues in AFM 27

Fig. 21 Illustration of the effects of sample drift. (a) This height image was
obtained with a sample (a monolayer on a gold film) that was not at thermal
equilibrium. The features are stretched, and imaging unstable. (b) The height
image on the right was obtained under exactly the same conditions, after the
sample had cooled to reach thermal equilibrium

Finally, thermal drift in AFM can occur both vertically as well as

horizontally. While AFM samples tend to be planar, and thus
expand mostly in the x–y plane, significant shift can occur in the
z axis as well. This is due to the sample-instrument planar misalign-
ment. The practical implication of this is that the sample can drift
out of scanner z-range during scanning if thermal drift is taking
place.
If thermal drift is suspected, the best ways to overcome it are to
scan more quickly, or to allow the sample time to come into thermal
equilibrium, as illustrated in Fig. 21.

3 Conclusions

There are a fairly wide range of artifacts that can be encountered in

AFM. The most common are artifacts stemming from probe dam-
age, which can be frustrating and time consuming, but are easily
diagnosed and rectified. The other artifacts discussed here are
generally rare occurrences. Knowing how to recognize and avoid
these artifacts makes AFM imaging considerably easier and more
productive.

Acknowledgments

This work was financially supported by UCIBIO/REQUIMTE via

grant UID/MULTI/04378/2013—POCI/01/0145/FERDER/
007728 from FCT/MEC through national funds and co-financed
by FEDER, under the Partnership Agreement PT2020.
28 Peter Eaton and Krystallenia Batziou
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