Atomic force microscopy

Mady by: Kristian Blom

Student number: 4359364
In 1985 three physicists, Gerd Binnig, Calvin F. Quate, and Christoph Gerber came up with an
incredible invention: The atomic force microscope (AFM). Atomic force microscopy is a scanning probe
microscope technique. Whereas with optical microscopes a sample is analyzed by detecting photons
which are emitted by the sample, with AFM a sample is analyzed by scanning the surface with a very
sharp tip. This scanning technique allows us to resolve surface details down to the atomic level
without damaging the probe nor the surface of the sample. Although scanning probe microscopes
already existed since 1981, the AFM had a huge advantage compared with scanning tunneling
microscope (STM), since it can image nonconducting surfaces.
This document is organized in the following way: First I will discuss the basic setup of the AFM system,
how to calibrate the AFM and the different imaging modes. Then I will discuss some of the limitations
of AFM and some of the biological applications of this technique. I will end this document with a
recap of the AFM hands-on activity.
AFM: Basic set-up, calibration of the AFM and imaging modes
With AFM a sample is analyzed by scanning the surface of that sample with a very sharp tip, usually
made of a small fractured diamond fragment. This tip is attached to a cantilever on which a laser
beam is reflected onto a position sensitive photo detector. The force between tip and sample is
calculated by measuring the deflection of the lever. Using Hookes law
=
it is possible to determine the force (F) when knowing the stiffness of the lever ( ) and the distance
(z) that the lever is bent.
In figure 1 a schematic view of a
typical AFM system is shown. The
colored dots represent different
parts of the set-up. Please notice
that an AFM can look very similar
to an STM. The most important
difference in the set-up is that
with AFM the sample moves
rather than the force sensor itself.
A micro-cantilever is attached
to a Piezoelectric (PZT) actuator.
Figure 1 - A schematic view of a typical AFM system (left), real micro-cantilever
Important to know is that a PZT
and components (right).
moves by mechanical strain
Jalili N, Laxminarayana K. A review of atomic force microscopy imaging systems: application to
resulting from an applied electric
molecular metrology and biological sciences. Mechatronics, Volume 14, Issue 8, 2004, 907945.
field. So, with the presence of an
external electric field, the sample will move by bending of the PZT.
A position sensitive quadrant photo detector, receiving a reflected laser beam off a mirror
mounted on the back of the cantilever ( ), makes it possible to detect movement of the cantilever

by measuring extremely small changes in the position of the light beam. These extremely small
changes result in a change of the photocurrents in the four distinct photosensitive elements, which
can be translated back to movement of the cantilever.
A feedback system, using the laser deflection, is used to make sure that the cantilever tip keeps it
z-position with respect to the sample surface at the desired height.
Now that we have discussed the basic setup of a typical AFM system, I will briefly explain how an
AFM can be calibrated. For this explanation I used the calibration method reported by Gibson et. Al.
(1)
Before using an AFM you need to know the spring constant of the cantilever. To determine this
constant, you need to have knowledge about the Youngs modulus and density of the cantilever.
Unfortunately, with fabrication of the cantilevers, there can be a 50% uncertainty of the thickness
quoted by the manufacturers. Also, since most cantilevers are coated with a thin gold layer (10-50
nm), the density can be 30% higher than indicated by the manufacturers. Therefore you need to
approximate both the density and thickness of the cantilever:
= 1 + /

(1)

~ + 1

(2)

:
1/ : /
:

These approximations are used to get the following expression:

3 (1 + ) = 0

1/ : /

(3)

:
:

Equation 3 can be solved for t, and then you can finally calculate
the spring constant using the following formula:

:
:

= (1 +

)(21.04)2

(4)

Notice that I dont show the formula to calculate , since this is a

rather long formula. The expression of F can be found in the article of Gibson et. Al (1) on page 114.
So, here we see that the spring constant can be determined by calibrating a cantilever using the
theoretical method above. Gibson et. Al have shown that this method works effectively for
determining the spring constant of an Ultralever. (1)
Once the AFM is calibrated, it can be used to determine the surface structure of all kinds of samples.
To measure different properties of the surface structure, different imaging modes are used. Here I
will discuss two of these imaging modes.
Contact mode:
Here the cantilever tip remains in contact with the sample
surface, much like the playback stylus of a phonograph
touches the surface of a record. For this imaging-mode there
are two different methods: constant force or constant height.
With the former the force exerted on the sample surface by
the tip is kept constant. With the latter the height of the tip
relative to the sample surface is kept constant. For this
method it is important that the sample surface is relatively

Figure 2 - Schematic representation of the

contact mode.
AFM slides course Nanotechnology (NB2081)

flat, otherwise the feedback cannot maintain control during scanning. This mode can sense the
repulsive forces between the tip and the sample.
Dynamic modes:
Non-contact mode: Here the cantilever tip is slightly above the sample surface and it is oscillating at
its resonant frequency. Of course it depends on the material used, but usually the resonant
frequency of the cantilever is about 1kHz. Microcantilevers made of silicon oxide are even lighter and
have resonant frequencies as high as 100 kHz. The higher the resonant frequency, the less sensitive
the cantilever is to vibrations, and the more stable it is for atomic force microscopy. This mode can
sense the attractive forces between the tip and the sample by mounting the cantilever on a PZT
element and measuring how much the frequency deviates from the resonance frequency. The bigger
the deviation the greater the attractive forces. The forces between the tip and sample are quite low,
on the order of 10 to 12 pN. To prevent the tip from coming to close to the sample surface, a
feedback system is used as indicated before.
Tapping mode: This mode is a combination of the contact
and non-contact mode, where the cantilever tip is oscillating
at its resonant frequency while the tip touches the sample
surface for a minimal amount of time. For this mode very
stiff cantilevers are used, since tips can get stuck in the
water contamination layer. The advantage of tapping the
surface relative to the contact mode is improved lateral
resolution for soft samples. Since the cantilever tip isnt
dragged over the surface in this mode, you dont suffer from
lateral forces such as drag.

Figure 3 - Schematic representation of the

tapping mode.
AFM slides course Nanotechnology (NB2081)

Please notice that there is another scanning

mode which is the electrical mode. Since this
mode isnt used as much as the modes
mentioned above, I wont discuss it.
Important to know is that for the three
different scanning modes, the measured
interatomic forces lie in a different domain.
This is represented in figure 2. Notice that
when the tip-sample separation becomes too
small, the sample surface will be damaged by
the sharp tip.
Figure 4 - The force measured vs. the distance between the
sample and the cantilever tip.
Jalili N, Laxminarayana K. A review of atomic force microscopy imaging systems:
application to molecular metrology and biological sciences. Mechatronics,
Volume 14, Issue 8, 2004, 907945.

Limitations of AFM and biological applications of AFM

Although the AFM is a great device for analyzing the surface of sample, it still has some limitations.
One disadvantage is that the AFM can only image a maximum height on the order of 10-20
micrometers and a maximum scanning area of about 150x150 micrometers. Another disadvantage is
the typical configuration of the cantilever tip, since this limits the xy-resolution. With typical
cantilever tips it is almost impossible to measure steeps walls or overhangs, due to the three

dimensional structure of the tip. An example of this will be given in the report of the hands-on
activity.
For biological purposes AFM is a great microscope technique to make three-dimensional images of
the structure of biological specimens. Not only is it possible to make images, but it is also possible to
interact with the sample by manipulating the biomolecules. Of course, the most important feature of
the AFM is that it is capable of monitoring biomolecular interactions. An example of a biological
application of AFM: In a recent study AFM was used to investigate the biological activity of Pa-MAP
1.9, which is a antimicrobial peptide derived from the polar fish Pleuronectes americanus that could
be used as a promising alternative to antibiotics. (2)
Description of the hands-on activity
On Tuesday 26-04-2016 I attended the AFM hands-on activity. The first thing to notice was that the
atomic force microscope was placed inside a soundproof closet, hereby the AFM is acoustically
isolated from its surroundings. Inside the closet we saw that the AFM-base was equipped with 4
hooks (every corner 1 hook) which in turn were connected to the closet ceiling using 4 elastic straps.
The advantage of these straps is that they have a very low resonance frequency, hence the AFM is
isolated from vibrations which come from the lab.
Next we talked about mica, which is a group of multilayer sheet minerals with a very soft and most
importantly very smooth surface. This characteristic makes mica very useful as subsurface for AFM,
since you want to have a clear distinction between the subsurface and the sample surface when
measuring with AFM.
Finally we observed the surface of a sample with the AFM. The sample which we observed were rings
of DNA, which is part of the so-called DNA origami. The rings were placed in a solution to which
magnesium was added. Since both DNA and mica have a negative charge, the positively charged
magnesium was needed to bind the DNA to mica (charge inversion). Once the AFM was turned on, an
image appeared on a computer screen layer by layer. The rings were clearly visible on the image. The
AFM measured that the holes inside the ring were 24 nm in diameter, but in real life they were 15
nm in diameter. This error was due to the convolution of the sample with the cantilever tip.
List of references
Articles:
1.
2.
3.
4.
5.

Gibson C T, et Al. (2003) Calibration of AFM cantilever spring constants. Ultramicroscopy 97: 113-118.
Cardoso M H, et Al. (2016) A polyalanine peptide derived from polar fish with anti-infectious activities.
Scientific Reports 6(21385).
Binnig G, Quate C F. (1985) Atomic force microscopy. Physical review letters 56(9): 930-934.
Hansma P K, Elings V B, Marti O, Bracker C E. (1988) Scanning tunneling microscopy and atomic force
microscopy: application to biology and technology. Science 242(4876): 209-216.
Jalili N, Laxminarayana K. (2004) A review of atomic force microscopy imaging systems: application to
molecular metrology and biological sciences. Mechatronics 14: 907-945.

Websites:
1.
2.
3.

http://www.nanoscience.com/technology/afm-technology/
http://machinemakers.typepad.com/machine-makers/2011/05/advantages-and-disadvantages-ofatomic-force-microscopy.html
http://www.nature.com/subjects/atomic-force-microscopy