This document provides an introduction to various types of microscopy used in nanotechnology. It explains that light microscopes have resolution limitations and then describes scanning electron microscopes which use electron beams rather than light to achieve higher resolution down to 1nm. Atomic force microscopes are also discussed, which use a physical probe to scan samples with nanoscale resolution. Finally, 3D optical profilers are introduced, which use light waves to generate 3D renderings of micrometer-scale surface topography. Overall, the document outlines different microscopy techniques and their applications from the micrometer to nanometer scale.

1. Getting a Closer Look:
An Introduction to
Nanotechnology Microscopy
By Kristina R. Boberg, NVCC
Image: http://jshs-tn.utk.edu/files/2012/10/Eye.jpg

2. Why do we use Microscopes?
• Sometimes we want to see
structures, surface details,
or even movement that
would be invisible to the
unaided eye.
• When dissecting, or
looking at a small sample,
we might just want a
magnified view.
• Or we want to see
specimens and organisms
at the tissue, cell, or
molecular level.
http://micro-scopic.tumblr.com/post/5551291228/anabaena

3. Scoping out the Competition
Until now, you’ve mostly
been using this type of
microscope,
but, as great as this scope
is, it has limitations. Now
you will learn about two
more types of microscopes
that will expand your
horizons, and allow you to
see beyond what light can
show you!
http://www.microscope.com

4. It’s all about
Resolution
Theoretically, an imaging
source (like a microscope)
should be able to resolve an
object the size of half the
wavelength of the imaging
energy.
So, if the object that you are
observing is smaller than half
the wavelength that you are
using, you won’t be able to
accurately resolve your
sample.
Structures that are close
together or that overlap
cannot resolve unless
the wavelength is short
enough.
http://www.ammrf.org.au/myscope/images/tem/resolution-wavelength.png

5. Will it Resolve?
http://www.nobelprize.org/educational/physics/microscopes/powerline/images/pl.gif

6. Move Away from the Light!
Light Microscope
• Wavelengths of Visible
light vary from 400-
700nm
• Light diffraction limits
resolution to 200-250nm
• Magnification limited to
around 1500x
Electron Microscope
• The wavelength of
electrons in an SEM is
5keV, or 0.017nm
• Resolution clarity to 1nm
• Magnification up to
500,000x
Versus

7. Essentially, instead of
light, you’re using
electrons emitted from a
source in the scope.
They’re focused through
a magnetic lens, and
interact with the
specimen to produce an
image based on the data
received from the
detectors.
So, What is a Scanning Electron
Microscope?
http://www.greenwood.wa.edu.au/resources/Physics%203B%20WestOne/content/
004_em_fields_force/images/pic066.jpg

8. How it WorksThe primary, or incident
beam scans the sample.
Some electrons “bounce”
off of the atoms in the
sample: those are
Backscattered electrons.
Some electrons are knocked
out of their energy levels by
the incident beam, and
these are Secondary
electrons—these are the
primary sources if specimen
information.
When a new electron jumps
energy levels to fill the hole
left by the incident beam,
energy is released as an X-
ray emission. http://www.ammrf.org.au/myscope/images/sem/volumes08.png
Surface of
Sample

9. What Can I Scan?
Since specimens are imaged in a
high-vacuum environment, there
are guidelines for what can be
scanned:
• The specimen has to be desiccated or
frozen (for cryogenic scanning).
• The specimen can not break apart or
outgas in the vacuum column.
• Ideally, it has to be conductive and
grounded (can be coated with a thin
layer of gold).
• It has to fit on the sample stub, and be
able to be mounted securely.
• Biological samples must be chemically
fixed, dried, and coated.
http://www.tedpella.com/SEMmod_html/16690.jpg
http://upload.wikimedia.org
http://isbbio1.pbworks.com/f/sem_8Group2.gif

10. This is What You Get
Large, clear images with
incredible detail.
K.Boberg,NVCC
K.Boberg,NVCC

11. But what if you want to look at something
that is even smaller, and perhaps still alive, or
is in liquid? Bacteria, living cells, proteins…
even DNA, perhaps?
(Sounds cool, right?
It is.)
You could use one
of these.
It’s an Atomic
Force Microscope.

12. What is Atomic Force Microscopy?
• AFM uses a physical probe to scan the sample,
instead of light or an electron beam.
• Nanoscale resolution—bonds between atoms
have been visualized on AFM, along with
viruses, DNA, and proteins.
• Samples don’t need to be in a vacuum, and do
not need to be conductive or desiccated
• Resolution more than 1000 times better than
the optical diffraction limit.

13. How does it Work?
A cantilever tip is put in contact with a
surface. An ionic repulsive force from
the surface, when applied to the tip,
bends the cantilever upwards.
The amount of bending is measured
by a laser spot reflected on to a
detector, and can be used to calculate
the ionic force.
Scanning the tip across the surface
allows the vertical movement of the
tip to follow the surface profile and is
recorded as the surface topography.
Here is a short video on how it works.
http://www.home.agilent.com/upload/cmc_
upload/ck/zz-other/images/AFM_schematic.gif

14. What You Can See*
* but only with optimum conditions and materials,
minimal vibration, a new cantilever, and with well-
prepared samples.
A living cell, Gold
Colloid, and DNA are
shown here.
Samples like these
can be viewed with
clarity and ease on
an AFM…

15. Back into the Light
We’ve been exploring probe
and electron microscopy, but
now it’s time to move back to
imaging instrumentation that
combines light and
technology to examine and
measure the topographical
features of a sample on a
slightly larger scale with a
3D Optical Profiler.

16. Optical Metrology Using the 3-D
Optical Profiler
Used for micrometer-scale
characterization, since the
wavelengths of light are
the limiting factor.
Provides a 3-D rendering of
the surface, to measure
the topographical features
of your sample.
Great for quality assurance
and measurements.
http://cwitechsales.com/Precision_Metrology.html

17. How does it work?
Optical profilers are used to measure height variations in sample
topography. These profilers use light waves to compare the optical path
difference between a sample and reference surface. This particular
profiler performs multiple Z-range scans in a defined area, and records
the XY locations and Z position of each pixel, and forms an image based
on this data. This allows what is termed “infinite focus”, meaning all of
the surface, regardless of height, is in focus at once in the final rendering.
http://www.zygo.com/?/met/profilers/opticalprofilersabout.htm

18. What samples can you use?
You can use anything that will fit under the objective!
Wet, dry, living or inorganic—the 3D Optical Profiler can
image it.
Flatter items are
preferable, to get the
most surface area in one
image, but insects,
plants, bacterial cultures,
and inorganic samples
can be measured and
characterized in full-
color detail.
http://www.zeta-inst.com/products/true-color-3D-optical-profiler

19. What do you see?
• Full-color, perfectly focused
images, and topographical
measurements of your sample.
http://www.nanoscience.com/products/optical-profilometry/optical-metrology-platform/

20. With Great (Magnifying) Power…
comes great responsibility.
These imaging instruments are very powerful, but
require a very delicate touch, constant care,
meticulous sample preparation, and frequent
calibration, which can be time consuming and
costly. That is why they are not as common as the
microscopes normally found in a classroom.
However, when you do have access to them, they
are an invaluable source of information and
scientific data as nanotechnology becomes more
important in the world of science and technology.

21. Thank you!
Now that you have all of this new information,
think of ways the instruments described and
demonstrated today can be incorporated into
your studies here at NOVA, and how they might
influence your future.
If you have any further questions, contact:
• Dr. Ia Gomez at igomez@nvcc.edu, or
• Kristina Boberg at kboberg@nvcc.edu.

22. Microscopy and Nanotechnology
Resources
• https://www.jic.ac.uk/microscopy/scale.html
• https://www.jic.ac.uk/microscopy/intro_LM.html
• https://www.jic.ac.uk/microscopy/intro_EM.html
• http://home.nas.net/~dbc/cic_hamilton/emicro.html
• http://nanotechweb.org/
• http://nano4me.org
• http://www.understandingnano.com/resources.html
• http://www.nanosurf.com/?content=04&gclid=CN7E24SyvsACFVQV7Aod
M3wAMQ