BIOL/MBIO/PBIO 5364 - Transmission Electron Microscopy

Principles and Techniques of Transmission Electron Microscopy

BIOL/MBIO/PBIO 5364 - Fall 2019

Instructional Staff:
George Lynn Cross Research Professor: Scott D. Russell, 210 Noble Microscopy Laboratory 405-325-4391 E-mail: srussell@ou.edu
Research Scientist: Preston Larson, 205E Noble Microscopy Laboratory 405-325-4391 E-mail: plarson@ou.edu

Instructional Schedule:
Lecture: 8:30 - 9:20 am Monday Wednesday Friday Laboratory: TBD
Microscopy hours: Four hours per week in addition to above

Web resources:

Introduction to the Course:
- Syllabus: Lectures, Labs and Readings
- Requirements: Micrographs, Projects, Focus Series (see syllabus)
- Safety: Precautions and Lab Procedures
- OU's Canvas - lectures, other handouts, announcements, student email
Biological Specimen Preparation:
- Lecture Material
  - Theoretical Basis - a discussion of fixatives, dehydration, infiltration, embedding
  - Toxicity of EM Chemicals
  - Safety Examination
- Detailed Information and Comparison of Preparation Techniques
  - Buffers
  - Fixatives
  - Dehydration
  - How to Handle Specimens
  - Embedding Media
  - Perfusion - specialized technique for animals
  - Generalized Schedule
- Sectioning
  - Thick sectioning - preliminary block preparation
  - Thin sectioning - ultramicrotomy
  - Making glass knives - how to use the LKB Knifemaker
  - LKB Knifemaker 7800B - diagrams and detailed drawings
- Online Calculators (uses JavaScript):
  - Calculate Fixatives and Buffers for one fixative, or calculators for:
    - for two fixatives
    - for three fixatives
  - Calculate Molarity
  - Calculate Percent
- Fixative Formulary:
- Buffer Formulary:
  - Cacodylate Buffer, 0.2 M
  - Maleate Buffer, 0.05 M
  - Ding Buffer - sucrose based cryoprotectant
- Stain Formulary:
  - Sato's Lead (WORD) | Sato's Lead (PDF)
  - Periodic Acid-Thiocarbohydrazide-Silver Proteinate Reaction
- Protocols:
  - Chemical Fixation:
  - Physical Fixation:
    - Spray-Freezing-Freeze Substitution of Unicellular Organisms for TEM
    - Protocol developed at OU for SF-FS: Fields, S. D., G.W. Strout, and S. D. Russell. 1997. Spray-freezing freeze substitution (SFFS) of cell suspensions for improved preservation of ultrastructure. Microscopy Research and Technique 38: 315-328 [Size: 1.1MB]
  - Other Protocols:
    - Silane - adheres cells to microscope slides (very sticky)
    - A freeze-fracture protocol from OU: Southworth, D., G. W. Strout and S. D. Russell. 1997. Freeze fracture of Plumbago zeylanica L. sperm in pollen and in vitro. Sexual Plant Reproduction 10: 217-226 [Size: 6.1MB]
    - Calcium localization protocol from OU: Tian HQ, Zhu H, Russell SD. 2000. Developmental changes in calcium distribution and accumulation in fertilized and unfertilized ovules and embryo sacs of Plumbago zeylanica. Sexual Plant Reproduction 13: 11-20. [Size: 753KB]
- Datasheets:
  - Blank Datasheets
  - A Sample Dataset for Pollen
Introduction to the Transmission Electron Microscope:
- Mechanical:
  - Vacuum Pumps and Systems
- Electronic:
  - Electron Beam Generation
  - Electron Sources
- Electron Optics and Resolution:
  - Limitations to Resolution (diffraction limited: Abbe's Equation)
  - Electron Optics and Aberrations
- Online Calculator (uses JavaScript):
  - Effects of Accelerating Voltage
Energy-Dispersive X-Ray Analysis
Histochemistry and Cytochemistry
- Periodic Acid-Thiocarbohydrazide-Silver Proteinate Reaction
Stereological Analysis
- Stereology Error Calculator
- Error Calculations
- John Russ's Practical Stereology - online PDF file of the second edition of his book [Size: 7.1MB]
Class Activities
- Trouble-shooting TEM
Miscellaneous
- Formulation of EM film photographic developer