BIO SCI 204-544 SYLLABUS
TRANSMISSION ELECTRON MICROSCOPY LABORATORY
SPRING 2011
Instructor:
Office:
Office Phone:
Lab Phone:
Home Phone:
e-mail
Heather Owen
Lapham 397
229-6816
229-4186
963-4826
owenha@uwm.edu
This course will train you to operate the Hitachi H-600 Transmission Electron Microscope (TEM) located in
the Department of Biological Sciences EM Lab in conventional mode, and in the basic techniques of
biological specimen preparation (conventional chemical fixation, dehydration, epoxy resin embedding,
ultramicrotomy, positive and negative staining). You will also learn techniques required for production of
transmission electron micrographs and how to prepare plates of digital images suitable for publication.
Because of the large amount of individual instruction that you will require to become skilled in these
techniques, your attendance at every laboratory session is vital. Be aware that to successfully complete
this course you will need to spend additional time outside of the regularly scheduled class meetings
working in the laboratory. Please note that failure to complete projects or submit work by the deadlines
listed in this syllabus will result in the deduction of points from your grade.
The required textbook for this course is:
Electron Microscopy: Principles and Techniques for Biologists by John J. Bozzola and Lonnie D.
Russell, 2nd Edition (Jones and Bartlett Publishers). This book is available at Panther Books.
In the event of disruption of normal classroom activities due to an H1N1 swine flu outbreak, the format for
this course may be modified to enable completion of the course. In that event, you will be provided an
addendum to this syllabus that will supersede this version. Information on UWM policies and procedures
related to exams, incomplete grades, religious holidays, discriminatory conduct, student misconduct,
grade appeals, and students with disabilities can be found at: www.uwm.edu/Dept/SecU/SyllabusLinks.pdf
COURSE EVALUATION
Project Proposal
Block Face and Semi-thin Section Check-off
Ultrathin Section and Staining Check-off
Alignment Check-off
Proficiency Project
20% of Final grade
10% of Final grade
10% of Final grade
10% of Final grade
50% of Final grade
PROJECT PROPOSAL
The Project Proposal is due at the beginning of class on March 11. This proposal should be written in the
style of a typical research paper, consisting of the following sections:
Title - must be descriptive of the work to be done.
Introduction - a description of your research project that includes an appropriate literature survey
(cited properly in the text according to requirements of the journal you have chosen as most
appropriate for publication of work in your field - see boldface below) and the significance of your
project.
Materials and Methods - describe how your specimens will be grown, collected or acquired, the
method of specimen preparation that will be used (include a flow chart of steps in specimen
preparation), and a description of the images you plan to collect. Information about the
manufacturers of equipment used must be included in this section.
Literature Cited - a list of all of the references you have cited in the proposal, formatted according
to the requirequirements of the journal you have chosen as most appropriate for publication of
work in your field - see boldface below.
Your Proposal should not exceed 10 pages (including citations). Double-space the body of the proposal
(Literature Cited can be single spaced, with a space between entries). The way references are cited in
the body of the text and the way references are formatted in the Literature Cited section MUST be
in the style of a journal that would be appropriate for the publication of your work. Include a copy
of the instructions for contributors from the journal you choose with your proposal. Most
publishers provide this information on their websites. If your project will involve the use of vertebrate
animals, a copy of the UWM Animal Care and Use Committee Protocol Form must be included with your
proposal. Before any animal experimentation is initiated, you must have an approved protocol on file. The
proposal will be edited and returned to you for inclusion in the Proficiency Project (it will make up portions
of your Introduction, Materials and Methods, and Literature Cited sections).
Thirty percent of your grade will be determined by your performance on three different "check-off"
assignments. Directions for each of these follow.
BLOCK FACE AND SEMI-THIN SECTION CHECK-OFF
This first check-off must be completed by March 17. To pass this check-off, you must produce an
appropriately trimmed block face that I will examine. The block face must be of the appropriate size and
shape for ultramicrotomy, and the block face must have been polished to a mirror finish by ultramicrotomy
with a glass knife. A microscope slide containing stained semi-thin sections that you have cut from the
block will also be examined at this time. Any specimen block is appropriate; it does not have to be a
specimen that you have prepared.
ULTRATHIN SECTION AND STAINING CHECK-OFF
To pass the second check-off (Section and Staining Check-off), you must produce a grid containing
stained ultrathin sections that I will examine with the TEM by April 14. For this check-off, sections can
be taken from any specimen block containing tissue. They do not need to be from specimens that
you have prepared yourself.
ALIGNMENT CHECK-OFF
The third check-off, the TEM Alignment Check-off, will require you to insert a specimen that I give you,
align the instrument and obtain a focused image of the specimen without using any written instructions. I
will check your image and microscope alignment, then ask you to answer some questions to demonstrate
that you are sufficiently skilled to operate the instrument correctly. Until you pass this check-off, you are
only authorized to use the transmission electron microscope when I am on campus. You may ask to
perform this check-off whenever you feel you are ready, but it must be passed by April 21.
PROFICIENCY PROJECT
The completed Proficiency Project is a research paper written as a journal article and will consist of the
following sections: Title, Abstract, Introduction, Materials and Methods, Results (must include a minimum
of 3 labeled plates of transmission electron micrographs with descriptive figure legends), Discussion, and
Literature Cited. Please note that each plate must consist of multiple micrographs (i.e. more than
one), assembled according to the specifications for image plates given in the instructions for
contributors from the journal that you chose when you submitted your project proposal. You will
also need to turn in study prints for all of the negatives that are used in your project. The Proficiency
Project is due by 5:00 p.m. on May 20, 2011.
CLASS SCHEDULE
Date
Week 1
Week 2
Week 3
Week 4
Week 5
Week 6
Topics
Jan 25
Lab Orientation, Key and Locker Assignment, Safety Discussion,
Hair Tool Preparation, Block Face Trimming
Jan 27
Preparation of Glass Knives
Feb 1
Cleaning Grids, Preparation of Support Films
Feb 3
Ultramicrotomy Training
Feb 8
Ultramicrotomy (continued)
Feb 10
Introduction to TEM - Specimen Insertion
Feb 15
Introduction to TEM - Instrument Alignment I
Feb 17
Introduction to TEM - Instrument Alignment II
Feb 22
Introduction to TEM - Instrument Alignment III
Feb 24
Individual Project Discussions
Individual Practice of Techniques
Mar 1
Preparation for Specimen Fixation, Calculations,
Fixative Preparation, Dehydration
Mar 3
Epoxy Resin Preparation
Date
Week 7
Week 8
Topics
Mar 8
Preparation of Lead Citrate and
Uranyl Acetate Stains
Mar 10
Positive and Negative Staining
PROJECT PROPOSALS DUE
Mar 15
Taking Micrographs with the TEM
Capturing Images for Magnification Calibration
Mar 17
BLOCK FACE AND SEMI-THIN SECTION CHECK-OFF DEADLINE
SPRING BREAK
Week 9
Week 10
Week 11
Week 12
Weeks 13 - 15
Mar 29
Removing and Developing TEM Film
Mar 31
Magnification Calibration
April 5
Printing Enlargements, Scanning Negatives
April 7
Assembling Plates in Photoshop
April 12
Writing Results & Figure Legends
April 14
SECTION AND STAINING CHECK-OFF DEADLINE
April 19
TEM ALIGNMENT CHECK-OFF
April 21
TEM ALIGNMENT CHECK-OFF
Work on Proficiency Project
PROFICIENCY PROJECTS MUST BE TURNED IN BY
5:00 P.M. FRIDAY MAY 20, 2011