Course Title: Laboratory Techniques in Electron Microscopy

Course Syllabus Fall 2014

Course Title: Laboratory Techniques in Electron Microscopy BIO 4001, 5001; 4003; 5003 Lecture Room and Time: LSE 207 1:00-1:50 pm MW; LSW Electron Microscope Facility Instructor: Dr. Stan Trauth Office: LSW 146 (Electron Microscope Facility); Office Hrs.: MTWTh 3-4 pm Textbook: Bozzola, J. J. and L. D. Russell. 1999. Electron Microscopy: Principles and Techniques for Biologists. 2nd Edition. Jones and Bartlett Publ., Sudbury, MA (ISBN: 0-7637- 0192-0) Objectives of Course: This course is designed to introduce upper level undergraduate and graduate students to the preparative techniques used in electron microscopy. In addition, the course will encourage research applications of the electron microscope. “Students with Special Instructional Needs

If you have any special needs related to learning or testing in this course, please let me know as soon as possible so I can address those needs.”

COURSE BOOK* AND OTHER USEFUL REFERENCES: *Bozzola, J. J. and L. D. Russell. 1999. Electron Microscopy: Principles and Techniques for Biologists. 2nd Edition. Jones and Bartlett Publ., Sudbury, MA (ISBN: 0-7637-0192-0) Dawes, Clinton J. 1988. Introduction to Biological Electron Microscopy: Theory and Techniques. Burlington, Vermont: Ladd Research Industries, Inc. Gabriel, Barbra L. 1982. Biological Electron Microscopy. New York: Van Nostrand Reinhold Co. (ISBN 0-442-22923-2) Gabriel, Barbra L. 1982. Biological Scanning Electron Microscopy. New York: Van Nostrand Reinhold Co. (ISBN 0-442-22922-4) Hayat, M. A. 1989. Principles and Techniques of Electron Microscopy. Boca Raton, Florida: CRC Press, Inc. (ISBN 0-8493-7111-2) Meek, Geoffrey A. 1976. Practical Electron Microscopy for Biologists. New York: John Wiley & Sons. (ISBN 0-471-99592-4) Weakley, Brenda S. 1981. A Beginner's Handbook in Biological Transmission Electron Microscopy. Edinburgh, England: Churchill Livingstone. (ISBN 0-443-02091-4) MAJOR GOALS: A. Course Goals: The ultrastructure student will: 1. describe and discuss standard preparative techniques used in electron microscopy. 2. recommend protocol schedules for preparation of biological specimens 3. analyze plastic-embedded materials using both light (thick sections) and electron ultrathin sections) microscopy 4. apply the use of the electron microscope in resolving various research problems 5. recognize the utility of the electron microscope as a valuable research tool in today's science, medicine and industry B. Specific Instructional Objectives: 1. fixation of tissues of choice 2. embedding of tissues in plastic 3. cutting of specimen blocks 4. staining tissues on copper grids 5. examining tissues with the electron microscope 6. photograph tissue

COURSE OUTLINE: A. Electron Microscopy 1. history and purpose of electron microscopy 2. construction and use of TEM 3. construction and use of SEM 4. preparation of specimens for electron microscopy B. Chemical Fixation for TEM 1. effects of chemical fixation 2. factors affecting chemical fixation 3. fixation procedures 4. types of chemical fixatives a. Osmium tetroxide b. Glutaraldehyde c. Formaldehyde d. Permanganates C. Preparation of Material for Embedding 1. handling tissue 2. rinsing 3. dehydration 4. water soluble plastics as dehydrants 5. dehydration schedules a. Ethanol - acetone b. Acetone D. Embedding 1. handling of plastics 2. infiltration 3. capsule embedding 4. flat embedding 5. particulate specimen embedding 6. embedding of delicate specimens E. Plastics 1. methacrylate (acrylic resins); plastic mixture 2. epoxy resins - (epon and araldite resins)

2 3. hardeners - (dodecenyl succinic anhydride, DDSA) 4. plasticizers - (dibutyl phthalate) 5. accelerators (catalysts) 6. curing mechanisms of anydride - epoxy systems 7. water soluble plastics F. Block Trimming and Knives 1. block trimming - (initial and final trim) 2. knives a. metal knives b. diamond knives c. glass knives

1st Technical Exam G. Sectioning and the Ultramicrotome 1. ultrathin sectioning 2. the ultramicrotome 3. sectioning 4. handling and storing sections on grids H. Staining 1. specificity of staining 2. stain procedures 3. lead stains 4. uranyl stains 5. phosphotungstic acid stains 6. permanganate stains 7. osmium staining 8. silver staining 9. ruthenium red staining I. Carbon and Metal Coating 1. high vacuum evaporatory 2. carbon evaporation 3. coating for SEM J. Thick Sectioning and Grid Preparation 1. thick section staining 2. types of specimen grids 3. grid cleaning K. Physical Techniques 1. critical point drying L. Preparative Techniques for Scanning Electron Microscopy 1. choice of accelerating voltage 2. preparation of biological specimens for SEM

2nd Technical Exam

COURSE REQUIREMENTS AND EVALUATION PROCEDURES: A. Attendance Policy: Mandatory attendance is required for both lecture and laboratory B. Handicap Policy: Any student having a certified handicapping condition will be allowed to make course modifications early in the semester. C. Examinations, Homework Assignments, and Presentations: 1. Memorizing course subject matter will not be sufficient to receive full credit on exams. This course requires that a student not only understand information, but also formulate and integrate facts and concepts into

3 answering subjective questions. 2. Preparation Tests will contain approximately 80% objective material and 20% interpretation of facts. 3. There will be two 100 point preparation exams. Preparation of labeled electron micrographs will be worth 100 points (approximately 5 micrographs will be graded). Class special projects will be worth 100 points. D. Make Up Policy: You must have an excused absence to make up work missed. Lecture exams must be made up within one week after the student has returned to class. E. Grading Scale: A = 100 - 90% B = 89 - 80% C = 79 - 70% D = 69 - 60% F = Below 60% F. Class Routine: The majority of in-class time will be lecture presentation of preparatory material. At the beginning of each lecture, a brief question-and-answer session will be conducted relevant to the topic at hand. G. Graduate Student Assignments: All graduate students are responsible for an extra assignment to be determined within 2 weeks following the beginning of the course.