MSE 360 Materials Laboratory I- Fall 2010

Microscopy and Microstructure Analysis

I. Objectives
Microstructure analysis is a core technique in Materials Science. Here we will introduce
the techniques of optical microscopy and scanning electron microscopy, and use these
instruments for the analysis of the microstructure for a cast 319 aluminum alloy that is
commonly used for engine blocks and heads in automobiles.

Over the three weeks of this laboratory we expect students to:

1. Develop skills and understanding of quantitative microstructural analysis by manual

statistical methods defined by appropriate ASTM Standards.
2. Perform statistical analysis of aggregate data (where possible) to determine standard
deviation, confidence levels and accuracy.
3. Become proficient in the use of the VVUL Nikon Optiphot microscopes and in the
basic theory of optical microscopy, including a working knowledge of standard optical
components and their functions, including digital image capture.
4. Acquire the necessary skills required for bulk specimen preparation, metallographic
polishing and etching to reveal microstructure.
5. Acquire a working knowledge of Scanning Electron Microscopy and the operation of
the VVUL SEM, and microstructure interpretation using SEM.
6. Be able to use automated digital image analysis techniques (Image J) for quantitative
microstructural analysis.
7. Skill development in constructing technical reports, including proper presentation of
images and graphs.
8. Demonstrate an understanding of phase diagrams in binary metallic systems and how
to use them in the interpretation of microstructure, particularly with respect to the role of
processing on microstructure.
9. Develop the practice of regularly using the

The emphasis will be on the proper preparation of a sample, the proper techniques for
acquiring an image, and the interpretation of features in the image.

II. Experimental Procedures

Laboratory I will be organized into several independent tasks to be conducted by
assigned groups. The order of tasks for each group will be assigned by instructors to
maximize use of facilities.

Activity 1: Metallographic specimen preparation. The use of the tools and instruments
for sectioning, mounting and grinding and polishing metal samples for subsequent
microstructural analysis by optical and scanning electron microscopy will be reviewed
and practiced.
Activity 2: Optical Microscopy. A GSI led review of the functions and operating
procedures of the Nikon Optiphot microscopes for reflected and transmitted light
microscopy. These microscopes will be used in the reflected light mode to obtain
images of metallographically prepared samples of Al 319.

Activity 3: These images will be used for quantitative analysis of microstructure, using
manual methods and/or Image J image analysis software. Each group will prepare three
samples removed from different sections of a laboratory casting of Al 319, identify
phases and constituent microstructures and compare changes in microstructures with
casting location.

Activity 4: The Philips XL30 Scanning Electron Microscope will be used for
microstructure analysis and for chemical analysis of phases by x-ray microanalysis using
the EDAX for Energy Dispersive Spectroscopy.

Specific instructions and reference materials for Laboratory I will be posted as resources
on CTools.

III. Background
Please review the optics you learned in physics, so you will be familiar with light and
lenses. For details on optical microscopy, you can consult the excellent “Microscopy
University” by Nikon, which can be found at: www.microscopyu.com. This is a good
resource. The “Reading for Lab 1” below is a good guide.

IV. Background References

The following references are suggested reading

1. C.A. Johnson, "Metallographic Sample Preparation", Metallography Principles and
Procedures, published by the Leco Corporation. (on MSE 360 Web Page)
2. E.E. Underwood, "Applications of Quantitative Metallography," Metals Handbook,
8th ed., vol. 8, p.37. (in 2235 HH Dow)
3. "Atlas of Microstructures of Industrial Alloys", Metals Handbook, 8th edition, vol. 7.
(in 2235 HH Dow)
4. “Metallography and Microstructures,” Metals Handbook Ninth Edition, Vol.9,
American Society for Metals, 1985. ISBN 0-87170-007-7 (In 2235 H. H. Dow)

Report
Following the format suggested in the Lab Manual, write a lab report that clearly
describes the objective of the experiment, the experimental results, and the conclusions
you can reach, based on your experimental data and observations. Provide all the
necessary data and appropriately reference the work you did to produce your data and
reach your conclusions (what page nos., appendices, graphs, etc.). Refer to the handouts
and lecture notes on report writing to help guide you in your efforts. As always, S.I. units
must be used lab reports, figures and tables. The lecture periods will cover the results
you should have and the expectations for the first laboratory report.

___________________________________________________________________

Image Analysis with Image J

There are many powerful image analysis software packages available. We will use an
open source program, Image J, available for free from the National Institute of Health
(NIH), which allows us to determine the area fraction of a phase, grain size, particle size,
etc. It is available on the Van Vlack computers, and on line from
http://rsbweb.nih.gov/ij/. There are hundreds of plug-ins for many analysis problems.
Most are biomedical (it is from NIH), but it is in common use by materials engineers.
We will use it to determine the volume fraction of the constituents in cast 319 Al and the
dendrite arm spacing as a function of position in the casting.

For quantitative analysis of volume fraction, you first have to process your raw image.
You must start with a good image, which after thresholding will clearly show the
constituent you want to measure. Please be aware that our human minds are very good for
recognizing images, but computers are very dumb. We can look at an image with large
grey dendrites and see only the dendrites, because our minds filter out the scratches,
blobs, pores, dirt, and extraneous junk. The computer only looks at pixels at the specified
greyscale – so it cannot tell meaningless junk from meaningful features, it cannot
distinguish a scratch from a grain boundary. So processing your image is important.

Make sure you have a good field of view one that shows what you want in the appropriate
size scale (appropriate magnification), with a representative view. When you characterize
the microstructure of a macroscopic object from a 500x micrograph, you are presuming
that the 0.2 mm x 0.2mm field of view is representative of the whole object. The images
have to be representative of the material for valid quantitative analysis. Avoid bias!
Everyone wants to take an image of a pretty area, so a collection of micrographs tends to
overemphasize the “nice” parts of a specimen, not the typical parts. (Similarly, postcards
of the United States are more likely to show Yosemite National Park than a northern
Ohio cornfield.)

Once you choose your image, you should change it from a color RGB image to an 8-bit
greyscale image. Do that with the drop-down menu Image>Type>8 bit. Next you want
to enhance contrast. Use the command Process>Enhance Contrast and select “OK” to
get the desired saturated pixel values (start at 0.5). Look at your image to make sure that
artifacts are not being emphasized (eg. scratches). Now you want to Normalize the
image, or recalculate the pixel values so the range of the pixels is equal to the maximum
range. Go to Process>Enhanced Contrast and select the “normalize” box and then “OK”.

Now you want to find the edges of your features (say the proeutectic microconstituent).
The software does this by looking for the spatial gradient at each point on the image. Use
the command Process>Find Edges. This might produce some spurious noise, which
must be removed by thresholding. Thresholding converts the greyscale image to black
and white. To do that, use the command Process>Binary>Threshold. Notice what this
does: it throws away all the information in the image except black pixel/white pixel. If
you are quantifiying proeutectic features in Cu-P, then everything about your image of
has now been reduced to “is not proeutectic” and “is proeutectic”. Let’s hope that
everything that is black is correctly proeutectic… or else we have an error.

You can now analyze the particles, counting the number, size and volume fraction of the
black (or white) features. Do that with the command Analyze>Analyze Particles. The
software will generate histograms, report averages, report statistics, etc. There are many
more commands and operations to do distinguish between several phases, separate
particles that touch, recognize grain boundaries, determine grain size, etc. ImageJ is
quite versatile and very powerful.

This is only a very brief introduction. Play with ImageJ and learn how to use it. Let it
give you USEFUL information about your microstructure. But do not let it deceive you
with NONESENSE. Notice how the “answer” varies from one field of view to another
(this is an indication of spatial heterogeneity). Notice how the “answer” changes with
details of thresholding. Different operators, choosing different saturated pixel values,
different image processing, different thresholding parameters, etc. will come up with
different “answers”. Your task is to find the correct answer – the volume fraction (or
grain size or whatever) that most effectively represents the microstructure of your
material. Do not be surprised if ImageJ reports 23.2148375% for one person/trial/field of
view and 17.49385937% for another person/trial/field of view. All it means is that the
volume fraction is about 20 +/- 3%. All these extraneous digits are meaningless noise.

Reading Assignments for Lab I

From: www.microscopyu.com

In the following list of topics from Nikon’s MicroscopyU site map, “X” indicates
required reading for MSE 360 Lab I: Microscopy, Digital Image Capture and
Quantitative Microscopy. In many of the sections you will find Java demonstrations of
key principles of microscopy and digital image capture, as well as excellent explanations
and illustrations. When you tire of reading, you may want to reward yourself by viewing
the movies of crystals in Part IV. Popcorn is recommended anywhere except the Van
Vlack Lab. I also encourage you to review the Nikon Small World Image Contest
website (Part V). In Part VI you will find a handy indexing of the interactive tutorials
embedded in many of the sections. You should be able to link directly to the sites from
the list using the Microsoft Word document, or you should go directly to the website and
use the list below as a guide.
 Nikon MicroscopyU Site Map
The MicroscopyU site map is designed to enhance navigation through the website using a
hierarchy of folders and direct links to articles and tutorials of interest. In order to explore
the site map, use the mouse cursor to expand folders having a plus (+) sign on the
directory tree. Blue text indicates a link, while black text is reserved for directory titles
(without a link). Folders can be collapsed by clicking on the minus (-) sign on the
directory tree adjacent to the folder name.

Nikon MicroscopyU
x I. Basic Concepts in Optical Microscopy
Conjugate Planes in Optical Microscopy
x Microscope Alignment for Köhler Illumination
x Depth of Field and Depth of Focus
x Field of View
x Refractive Index
x Numerical Aperture
x Resolution in Optical Microscopy
x Useful Magnification Range
x Working Distance and Parfocal Length
Image Brightness
Coverslip Correction
Adjustment of Objective Correction Collars
Linear Measurements (Micrometry)
Specimen Contrast in Optical Microscopy
x II. Microscope Optical Systems
x The Microscope Optical Train
Infinity Optical Systems
x Introduction to Microscope Objectives
x Properties of Microscope Objectives
x Microscope Objective Specifications
Specialized Microscope Objectives
Water Immersion Objectives
Nikon CFI60 Optical System
Modulation Transfer Function
Phase Contrast Microscopy
Differential Interference Contrast (DIC) Microscopy
Fluorescence Microscopy
Nikon Fluorescence Filter Sets
Live-Cell Imaging
Confocal Microscopy
Stereomicroscopy
x III. Digital Imaging in Optical Microscopy
Digital Imaging – New Opportunities for Microscopy
x Fundamentals of Digital Imaging
x Introduction to Charge-Coupled Devices (CCDs)
Color Balance in Digital Imaging
x Digital Camera Resolution Requirements for Optical Microscopy
x CCD Signal-To-Noise Ratio
Nikon Digital Camera Systems for Optical Microscopy
Principles and Applications of Interferometry
Miscellaneous Review Articles
Nikon's Museum of Microscopy
Digital Image Galleries
IV. Digital Video Galleries
x Chemical Crystals Movie Gallery
x Live Cell Imaging: Cell Motility
x Pond Life Movie Gallery
Nikon's Featured Microscopists
V. Nikon Small World Competition
Screensaver Software
x VI. Interactive Java and Flash Tutorials
MicroscopyU Knowledge Database
Copyright and Image Use Information
Use Microscopy U (or another resource) so that you become familiar with the following
terms. We might ask you about this in the lab, or we might have a little quiz.

Terms and Definitions for Optical Microscopy and Digital Image Capture
Optical Microscopy

1. Microscope Illumination (Kohler, alignment)

2. Condenser Aperture

3. Objective Aperture

4. Depth of Field

5. Index of Refraction (Snell’s Law)

6. Numerical Aperture

7. Field of View

8. Optical Dispersion (chromatic aberration)

9. Image Resolution (Airy Patterns)

10. Useful Magnification/Empty Magnification

11. Working Distance

12. Parfocal Length

13. Components of Optical Microscope (Nikon Optiphots in VV Lab)

14. Specifications on Objective Lenses

15. Polarizing Light for Optical Microscopy

Digital Image Capture

1. Charge Couple Device (device description, materials, principles)

2. Pixel

3. Quantum Efficiency

4. CCD Noise/Signal to Noise Ratio

5. CCD Array Size

6. Dynamic Range

7. Variables Controlling Image Capture Speed

8. Physics and Methodology of Image Readout

9. Resolution Requirements for Optical Microscopy