BIO SCI 204-544 SYLLABUS
TRANSMISSION ELECTRON MICROSCOPY LABORATORY
SPRING 2011
Instructor: Heather Owen
Office: Lapham 397
Office Phone: 229-6816
Lab Phone: 229-4186
Home Phone: 963-4826
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 20% of Final grade
Block Face and Semi-thin Section Check-off 10% of Final grade
Ultrathin Section and Staining Check-off 10% of Final grade
Alignment Check-off 10% of Final grade
Proficiency Project 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 Topics
Week 1 Jan 25 Lab Orientation, Key and Locker Assignment, Safety Discussion,
Hair Tool Preparation, Block Face Trimming
Jan 27 Preparation of Glass Knives
Week 2 Feb 1 Cleaning Grids, Preparation of Support Films
Feb 3 Ultramicrotomy Training
Week 3 Feb 8 Ultramicrotomy (continued)
Feb 10 Introduction to TEM - Specimen Insertion
Week 4 Feb 15 Introduction to TEM - Instrument Alignment I
Feb 17 Introduction to TEM - Instrument Alignment II
Week 5 Feb 22 Introduction to TEM - Instrument Alignment III
Feb 24 Individual Project Discussions
Individual Practice of Techniques
Week 6 Mar 1 Preparation for Specimen Fixation, Calculations,
Fixative Preparation, Dehydration
Mar 3 Epoxy Resin Preparation
Date Topics
Week 7 Mar 8 Preparation of Lead Citrate and
Uranyl Acetate Stains
Mar 10 Positive and Negative Staining
PROJECT PROPOSALS DUE
Week 8 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 Mar 29 Removing and Developing TEM Film
Mar 31 Magnification Calibration
Week 10 April 5 Printing Enlargements, Scanning Negatives
April 7 Assembling Plates in Photoshop
Week 11 April 12 Writing Results & Figure Legends
April 14 SECTION AND STAINING CHECK-OFF DEADLINE
Week 12 April 19 TEM ALIGNMENT CHECK-OFF
April 21 TEM ALIGNMENT CHECK-OFF
Weeks 13 - 15 Work on Proficiency Project
PROFICIENCY PROJECTS MUST BE TURNED IN BY
5:00 P.M. FRIDAY MAY 20, 2011