Design a Lab: Differentiate between cancerous and noncancerous cells using Atomic Force Microscopy.
Background Information
AFM (Atomic Force Microscopy) is one of the most widely used instruments in
nanotechnology. AFM is a tool used to image, measure and manipulate matter at the
nanoscale. In the early stages of AFM, it was used mostly to scan and understand what the
surfaces of various substance looked like at the nanoscale. However, by modifying the tip, we
can now measure so much more (electric and magnetic properties, chemical potentials,
friction etc), and perform various types of spectroscopy and analysis.
The function of the an AFM is based upon 3 elements:
1) An atomically sharp tip (probe) on the end of a flexible cantilever beam deflects in
proportion to the force of interaction;
2) A piezoelectric transducer to facilitate positioning and scanning the probe in three
dimensions over the sample with very precise movements; and
3) A feedback system to detect the interaction of the probe with the sample.
Figure 1: Atomic Force Microscope. Here we
have a covered table that floats on air. The air
cushion provides protection against vibrations,
while the metal cover prevents electrical
interference. (Photo taken at NINT at the
University of Alberta)
Figure 2: Under the cover of Figure 1:
The AFM instrument itself. The bright
silver box in the middle contains a
position-sensitive photodiode system
that tracks the location of the tip. The
cylinder immediately below this is a
piezoelectric material that changes
shape slightly with applied voltages.
This controls the location of a tip located
at the bottom of the cylinder, see figure
3. (Photo taken at NINT at the
University of Alberta).
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How AFM works:
A cantilever with a probe (aka. Tip), is used to scan a sample of material. The researcher uses
the probe to create a digital image of the topography of the sample (aerial view). It may also
be used to determine the thickness of material at the nanoscale.
To gain a better understanding of how to set up a sample for AFM, watch this video (from
0:00 to 2:47 minutes): https://www.youtube.com/watch?v=dhfrZF3kAoQ
Figure 3: The set-up of AFM. Note the uneven
sample, which would look and feel smooth to our
fingers, but is not so at the nanoscale. Block
diagram of atomic force microscope using beam
deflection detection. As the cantilever is
displaced via its interaction with the surface, so
too will the reflection of the laser beam be
displaced on the surface of the photodiode.
(http://en.wikipedia.org/wiki/Atomic_force_mic
roscopy)
The development of AFM, it is important to know that the original nanoscale microscope was
called STM (Scanning Tunneling Microscope), which worked well with conductive materials.
Atomic Force Microscopy was developed as a need to observe non-conductive materials,
although it can be used for conductive surfaces as well.
Watch this tutorial to better understand scanning probe microscopy:
http://virtual.itg.uiuc.edu/training/AFM_tutorial/
Figure 4: The AFM tip holder, which is how
a microscopic tip is loaded into the
instrument. The tip is located at the apex
of the holder on the left, and it is the small
line (barely visible) that a laser must be
directed onto in order to track the
position. (Photo taken at NINT at the
University of Alberta)
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Viewing thickness
Recall that AFM is used to determine the thickness of various materials, or establish how
smooth a surface is. For example, the surface of a glass table is smooth to our touch, but when
we look at that same piece of glass using AFM, it appears rough (see figure 5).
Figure 5: Clean glass viewed using AFM (image obtained from
http://www.eng.utah.edu/~lzang/images/Lecture_10_AFM.pdf
Nanotechnology researchers at NINT have determined that when any substances are at their
atomic level (single atom), the properties and behavior of those materials are completely
different than when they are molecules (groups of same atom). For example, the melting point
of any metal substance, such as gold, decreases significantly when a single atom is isolated.
About AFM Probes (aka AFM Tips)
The type of mode selected is completely dependent upon what type of information the
researcher is searching for. Along with the mode, the appropriate probe (or tip) must be
selected depending on the material being tested. There are specialized probes for all types of
materials. For example when observing glass: depending on the feature size you are
interested in, you would go with a general-purpose probe (lateral resolution >5nm), or a
sharp, high-resolution tip (to probe features down to 1 nm in size). If you were planning to
scan relatively large areas for a long time, you might consider wear-resistant tips to avoid
gradual changes in tip quality during the measurement; there are diamond- or diamondcoated tips available.
The images on page 4 & 5 were taken using a general purpose probe
Dead and live (and barely live) cells generally present identical requirements. You typically
want to study them in the liquid, as dried cells would not provide much relevant information
due to severe changes in properties. Therefore, you would choose the probes designed for
biological imaging (or other "soft" imaging) in fluid.
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A Real NINT trial:
The following chip (figure 7) was built by Nikola Pekas and was used to run tests by flowing
electrical current through these carbon and gold line intersections. Upon microscopic
inspection (Figure 8), damage to the area was noted, yet not visible to the naked eye. Using
AFM (Figure 9), Dr. Pekas was able to more closely analyze the damage to the chip: “The AFM
image zooms in further to reveal that edges of the damaged area feature small "balls",
indicating that gold was melted in the area (when melted, it tends to "bead up" into droplets,
and they form spheres when they cool down and solidify, just like wax dripping down the
candle forms little spherical bumps). It turns out that the damage was due to electrical
breakdown of the structure because the voltage we applied was too high - literally a spark
was formed that melted the gold layer. In other words, ‘we fried it’.”
Camera image
Figure 7: A camera view of a chip with intersecting lines made
of carbon and gold. Carbon lines are gray (just like graphite in
the pencil), gold lines are purple (they are only 30
nanometers thin).
Figure 8: The damaged area on the chip from
the electrical current flowing.
Microscope Image
Figure 9: The AFM image zoomed in. Dr. Pekas likely
used tapping mode (see page 5 for details)
Figure 10: AFM Cross Section
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There are 3 Modes of AFM Operation1
1. Contact Mode
Contact mode is used to test if the substance is slippery or sticky, by dragging the tip across
the surface. Contact mode may be used to pull away a layer of the material as the tip is
dragged to expose what might be below (this is called digging a trench), as seen in Figure 5.
ce Microscopy
3.5
% Points
2.5
tp://en.wikipedia.org/wiki/File:Atomic_force_microsc
e_block_diagram.svg
3.2 nm
AFM Histogram Data
Gaussian Fit x1
Gaussian Fit x2
3
2
1.5
1
0.5
0
20
Figure 5
3.5
Height (nm)
30
Figure 6
AFM Histogram Data
Gaussian Fit x1
Figure 5 & 6: AFM image of a
Gaussian Fit x2
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trench carved into a 3.2 nm thick molecular layer. This represents our
primary use of the instrument in contact mode- to determine and verify molecular layer thicknesses.
2.5
% Points
3.2 nm
25
2
1.5
2. Non Contact Mode
1
0.5
0
20
Non Contact mode is use when there is risk of damaging the sample when viewing. In this
mode, the tip oscillates above the surface of the sample using intermolecular forces for
repulsion or magnetism depending on the material being observed. This will also provide an
image of sample
topography, however
the resolution will be lower than in Contact mode.
25
30
Example of Non
Contact
Mode:
https://www.youtube.com/watch?v=Ha53tFTsmW8
.
Height (nm)
3. Tapping Mode
Tapping mode is used primarily to observe the topography of the surface. The AFM tip will
oscillate and tap the surface to create a digital, 3D image of the surface. This is the most
popular mode of AFM as it provides greater resolution than Non-Contact Mode, but poses less
damage to the material than Contact Mode. Example of Tapping Mode:
https://www.youtube.com/watch?v=mp7GC5pUtbQ.
Bonus Task: Build your own fully functional AFM Machine with LEGO:
Watch the video and use the parts list and instructions to build your own AFM system as
supported by the National Science Assembly:
http://mcise.uri.edu/park/MNEL/legoafm/index.html
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http://www.chembio.uoguelph.ca/educmat/chm729/afm/details.htm
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Your Task
You have been provided with information about the purpose of Atomic Force
Microscopy and how it works. Using the background information provided and
the internet, you will design a lab, involving AFM, to determine if there are
differences at the nanoscale to differentiate between healthy and cancerous
human tissue cells.
The following 3 papers provide some information as to the research being done
to determine the differences found at the nanoscale size, between cancerous and
healthy cells:
1. http://www.nature.com/nnano/journal/v4/n6/full/nnano.2009.77.html
2. http://www.ncbi.nlm.nih.gov/pubmed/22209753
3. http://www.nature.com/nnano/journal/v4/n2/full/nnano.2009.004.htm
l
4. http://www.nature.com/nnano/journal/v7/n11/full/nnano.2012.196.ht
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Your lab design must include:
 A problem statement,
 Hypothesis,
 Manipulated, Responding and Controlled variables,
 List of Materials
 Procedure (including the preparation of your sample and setting it up in
the AFM.
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