Practical Electron Microscopy (and other microscopies)
for the
Engineer, Scientist, and Artist
Course Number:
Instructor:
OPT 307/407
Brian McIntyre
Summary:
This is a performance-based course comprised of lectures to introduce concepts and labwork to
reinforce these concepts. The semester will begin with a historical overview of microscopic techniques followed by
a concentration in the Scanning Electron Microscopic (SEM) as well as other (SPM, LM, CLM, TEM) observation
of specimens. Micro-chemical analysis, in the context of the electron microscope, will also be discussed and
performed. Independent laboratory projects will be chosen by the students culminating in a poster and web-based
presentation of their results.
Grading:
Six lab reports 20%
Mid-term exam: 20%
Homework:
10%
Project:
(50% total)
Web presentation:
20%
Poster presentation:
30%
(Areas of concern: Format/Look, Clarity, Relevance, Difficulty, Completeness,
Timeliness)
(Include at least 5 of the following: SEI, BSE, LM, X-ray, Interaction modeling,
AFM/STM, Coating, CPD/HMDS, TEM/Diffraction, Microtomy)
Prerequisites:
Labs:
None
Calibration of the SEM
Sample Preparation: conductive samples, insulating samples
Biological Samples: critical point drying, HMDS
Imaging modes
X-ray spectrometry
(Graduate-level only) TEM and diffraction
Independent Projects
Lectures:
History, Fundamentals, Vacuum Technology, Electron sources, Electron lenses, Beam-Sample
interactions, Signal detectors, Scanning Probe (SPM) techniques, Atomic Force Microscope (AFM) demonstration
on optical materials and devices, Imaging, Sample preparation, Image artifacts, X-ray microanalysis, Quantification
of x-ray data
(Graduate-level only) Transmission electron microscopy and electron diffraction
Opt 307/407
Spring 2005
Course Syllabus
Week of
1: Jan 9
2: Jan 16
3: Jan 23
4: Jan 30
5: Feb 6
6: Feb 13
7: Feb 20
Lecture Topic
Introduction to Course
Text
Syllabus
Exams, Project, Lectures, Labs
Introduction to Microscopies
Vacuum Systems
Putting together your project proposal
Electron Sources and Lenses
The SEM System
Specimen Preparations
Tour of Pathology Lab at URMC
Image Recording
12: Mar 27
13: Apr 3
14: Apr 10
15: Apr 17
16: Apr 24
Complementary Techniques
The Art of Presentations
Artifacts and Advanced Topics
The TEM System (Graduate required)
Spring Break
SPM systems
Case Study (Organ Metal Project)
Light Microscopies/Confocal LM
Review/ Final Exam
Project Work
Project Work
Project Work
Project Work
17: Apr 28
Reading week/Poster presentation
8: Feb 27
9: Mar 6
10: Mar 13
11: Mar 20
mcintyre@optics.rochester.edu
x53058 Wilmot 206a (TEM lab)
x54875 Wilmot Annex (SEM lab)
Text
Lab
Chap 1-2
Chap. 14.1
Handouts
Chap. 14-15
Notes
Chap. 15
Chap. 16
Lab Tour
Vacuum (in-class). No report
due
1- Calibration check
2- Sample Prep.:
Conductors/Insulators
3- Sample Prep. Biologicals
Chap. 8,9,1617,19
Chap. 18
Handouts
Chap. 18
Handouts
4-Three imaging standards
Project proposals due
5- X-ray spectrometry
Handouts
Chap. 10
SPM demo (in class)
Approval of Projects
In-class photo critique
6-TEM imaging and diffraction
In-class discussion w/web and
poster
Wilmot display
History of the Microscope
Light Microscopes and Electron Microscopes
By Mary Bellis (adapted by Brian McIntyre 12/2003)
(Information from Newtom BBS A Division of Educational Programs at Argonne National
Laboratory and the National Institute of Health as well as web resources)
During that historic period known as the Renaissance, after the "dark" Middle Ages, there
occurred the inventions of printing, gunpowder and the mariner's compass, followed by the
discovery of America. Equally remarkable was the invention of the microscope: an instrument
that enables the human eye, by means of a lens or combinations of lenses, to observe enlarged
images of tiny objects. It made visible the fascinating details of worlds within worlds.
Long before, in the hazy unrecorded past, someone picked up a piece of transparent crystal
thicker in the middle than at the edges, looked through it, and discovered that it made things look
larger. Someone also found that such a crystal would focus the sun's rays and set fire to a piece
of parchment or cloth. Magnifiers and "burning glasses" or "magnifying glasses" are mentioned
in the writings of Seneca and Pliny the Elder, Roman philosophers during the first century A. D.,
but apparently they were not used much until the invention of spectacles, toward the end of the
13th century. They were named lenses because they are shaped like the seeds of a lentil.
The earliest simple microscope was merely a tube with a plate for the object at one end and, at
the other, a lens that gave a magnification less than ten diameters -- ten times the actual size.
These excited general wonder when used to view fleas or tiny creeping things and so were
dubbed "flea glasses. "
About 1590, two Dutch spectacle makers, Zaccharias Janssen and his son Hans, while
experimenting with several lenses in a tube, discovered that nearby objects appeared greatly
enlarged. That was the forerunner of the compound microscope and of the telescope. In 1609,
Galileo, father of modern physics and astronomy, heard of these early experiments, worked out
the principles of lenses, and made a much better instrument with a focusing device.
The father of microscopy, Anton Van Leeuwenhoek of Holland (1632-1723), started as an
apprentice in a dry goods store where magnifying glasses were used to count the threads in cloth.
Anton van Leeuwenhoek was inspired by the glasses used by drapers to inspect the quality of
cloth. He taught himself new methods for grinding and polishing tiny lenses of great curvature
which gave magnifications up to 270x diameters, the finest known at that time.
These lenses led to the building of Anton Van Leeuwenhoek's microscopes considered the first
practical microscopes, and the biological discoveries for which he is famous. Anton Van
Leeuwenhoek was the first to see and describe bacteria (1674), yeast plants, the teeming life in a
drop of water, and the circulation of blood corpuscles in capillaries. During a long life he used
his lenses to make pioneer studies on an extraordinary variety of things, both living and nonliving, and reported his findings in over a hundred letters to the Royal Society of England and the
French Academy.
"My work, which I've done for a long time, was not pursued in order to gain the praise I now
enjoy, but chiefly from a craving after knowledge, which I notice resides in me more than in
most other men. And therewithal, whenever I found out anything remarkable, I have thought it
my duty to put down my discovery on paper, so that all ingenious people might be informed
thereof." - Anton Van Leeuwenhoek Letter of June 12, 1716
None of Anton Van Leeuwenhoek's microscopes exist today. His instruments were made of gold
and silver and were sold by his family after he died.
Robert Hooke, the English father of microscopy, re-confirmed Anton van Leeuwenhoek's
discoveries of the existence of tiny living organisms in a drop of water. Hooke made a copy of
Leeuwenhoek's microscope and then improved upon his design. He looked at a sliver of cork
through a microscope lens and noticed some "pores" or "cells" in it. Robert Hooke believed the
cells had served as containers for the "noble juices" or "fibrous threads" of the once-living cork
tree. He thought these cells existed only in plants, since he and his scientific contemporaries had
observed the structures only in plant material.
Robert Hooke wrote Micrographia, the first book describing observations made through a
microscope. Hooke was the first person to use the word "cell" to identify microscopic structures
when he was describing cork. Hooke also wrote Hooke's Law -- a law of elasticity for solid
bodies.
Later, few major improvements were made until the middle of the 19th century. Then several
European countries began to manufacture fine optical equipment but none finer than the
marvelous instruments built by the American, Charles A. Spencer, and the industry he founded.
Present day instruments, changed but little, give magnifications up to 1250 diameters with
ordinary light and up to 5000 with blue light.
A light microscope, even one with perfect lenses and perfect illumination, simply cannot be used
to distinguish objects that are smaller than half the wavelength of light. White light has an
average wavelength of 0.55 micrometers, half of which is 0.275 micrometers. (One micrometer
is a thousandth of a millimeter, and there are about 25,000 micrometers to an inch. Micrometers
are also called microns.) Any two lines that are closer together than 0.275 micrometers will be
seen as a single line, and any object with a diameter smaller than 0.275 micrometers will be
invisible or, at best, show up as a blur. To see tiny particles under a microscope, scientists must
bypass light altogether and use a different sort of "illumination," one with a shorter wavelength.
The introduction of the electron microscope in the 1930's filled the bill. The invention of the
electron microscope was made possible by a number of theoretical and experimental advances in
physics and engineering. The main concept on which the electron microscope is founded—that
electrons have a wavelike nature—was hypothesized by French physicist Prince Louis Victor de
Broglie in 1923. In 1927, de Broglie’s hypothesis was experimentally verified by American
physicists Clinton J. Davisson and Lester H. Germer, and independently by English physicist
George Paget Thomson. Co-invented by Germans, Max Knott and Ernst Ruska in 1931, Ernst
Ruska was awarded half of the Nobel Prize for Physics in 1986 for his invention. (The other half
of the Nobel Prize was divided between Heinrich Rohrer and Gerd Binnig for the STM.) In this
kind of microscope, electrons are speeded up in a vacuum until their wavelength is extremely
short, only one hundred-thousandth that of white light. Beams of these fast-moving electrons are
focused on a cell sample and are absorbed or scattered by the cell's parts so as to form an image
on an electron-sensitive photographic plate. In 1938 Ruska and German engineer Bodo von
Borries built the first model of the commercial TEM for the Siemens-Halske Company in Berlin,
Germany. The English engineer Sir Charles Oatley invented the SEM in its present form in 1952.
Shown below is a similar microscope from the University of Toronto from 1938.
If pushed to the limit, electron microscopes can make it possible to view objects as small as the
diameter of an atom. Most electron microscopes used to study biological material can "see"
down to about 10 angstroms--an incredible feat, for although this does not make atoms visible, it
does allow researchers to distinguish individual molecules of biological importance. In effect, it
can magnify objects up to 1 million times. Nevertheless, all electron microscopes suffer from a
serious drawback. Since no living specimen can survive under their high vacuum, they cannot
show the ever-changing movements that characterize a living cell.
Using an instrument the size of his palm, Anton van Leeuwenhoek was able to study the
movements of one-celled organisms. Modern descendants of van Leeuwenhoek's light
microscope can be over 6 feet tall, but they continue to be indispensable to cell biologists
because, unlike electron microscopes, light microscopes enable the user to see living cells in
action. The primary challenge for light microscopists since van Leeuwenhoek's time has been to
enhance the contrast between pale cells and their paler surroundings so that cell structures and
movement can be seen more easily. To do this they have devised ingenious strategies involving
video cameras, polarized light, digitizing computers, and other techniques that are yielding vast
improvements in contrast, fueling a renaissance in light microscopy.
Relative Size Scales in the Macro and Micro (Nano) World
Development of Microscopic Imaging Techniques
Calibration of the SEM
Procedure:
Use certified latex microspheres and larger silicon calibration chip to calibrate over the 0.1 to
100 um range. Use built in measurement calipers to find sizes. Record micrographs with sizes
noted as image overlays.
Plot measured vs. certified size
Determine error and correction factor.
Data/Notes:
What you submit:
Name: ______________________
Date: ______________________
Report on:
________________
Materials and Methods:
Data:
Discussion:
Summary:
******************************************************************************