SEM, AFM, and 3D Optical Profiler Introduction

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

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

Scoping out the CompetitionUntil 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

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

Will it Resolve?

http://www.nobelprize.org/educational/physics/microscopes/powerline/images/pl.gif

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

Ve

rs

us

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

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

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.

htt

p:/

/ww

w.t

edp

ella

.co

m/S

EMm

od

_htm

l/16

690

.jpg

htt

p:/

/up

load

.wik

imed

ia.o

rg

http://isbbio1.pbworks.com/f/sem_8Group2.gif

This is What You Get

Large, clear images with incredible detail.

K. B

ob

erg,

NV

CC

K. B

ob

erg,

NV

CC

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.

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.

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

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…

Back into the LightWe’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.

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

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.

htt

p:/

/ww

w.z

ygo

.co

m/?

/met

/pro

file

rs/o

pti

calp

rofi

lers

abo

ut.

htm

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

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/

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.

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 [email protected], or

• Kristina Boberg at [email protected].

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=CN7E24SyvsACFVQV7AodM3wAMQ