Dr. Jeffrey M. Osborn

Dean of the School of Science and Professor of Biology
The College of New Jersey

Advanced Topics: Electron Microscopy - Biology 518

An in-depth study of selected science topics presented under formal classroom organization (not intended for individualized study). The total number of hours on a program is limited to 8; only those hours that have the approval of the student's advisor may be counted as biology electives.

1. Community Ecology
2. Electron Microscopy
3. Endocrinology
4. Eukaryotic Molecular Genetics
5. Microbial Ecology
6. On Human Nature
7. Parasitology
8. Plant Ecology
9. Virology

Course Syllabus

Course Information

Course Policies

1. Evaluations:
   Lecture

   Electron Microscopy (Biology 518) is principally a hands-on, technique-oriented class. As such, very few lectures will be presented and only one lecture exam will be taken. Specific readings, however, will be assigned, and students will be expected to demonstrate comprehension of the assigned material via incorporation into their lab work.

   Electron Microscopy is a course which employs active learning as its principal instructional method. Students will be interacting with the instructor, both individually and in small groups, in order to develop a series of technical skills. In addition to the fundamental techniques, students will learn some salient theoretical aspects of particular methodologies as well as develop the ability to interpret the results obtained through the various techniques mastered.

   To help ensure that students learn all techniques associated with the class, a series of deadlines have been set throughout the semester. For full credit, all deadlines must be met; there will be no penalty for fulfilling the various requirements before the deadlines. Furthermore, each student will be expected to pass a proficiency exam before she/he will be allowed to use any instrument without the instructor's supervision.

   The deadline and proficiency exam dates are listed on the course schedule (page 1). The details concerning individual course requirements and their respective point values are listed below.

   Microscopy is a course which employs active learning as its principal instructional method. Students will be interacting with the instructor, both individually and in small groups, in order to develop a series of technical skills. In addition to the fundamental techniques, students will learn a number of salient theoretical aspects of particular methodologies as well as develop the ability to interpret the morphological results obtained through the various techniques mastered.

   To help ensure that students learn all techniques associated with the class, a series of deadlines have been set throughout the semester. For full credit all deadlines must be met; there will be no penalty for fulfilling the various requirements before the deadlines. Furthermore, each student will be expected to pass a proficiency exam before she/he will be allowed to use any instrument without the instructor's supervision.

   The deadline and proficiency exam dates are listed on the course schedule (page 1). The details concerning individual course requirements and their respective point values are are listed below.

   Final Exams - The date of the lecture final is pre-scheduled by the University, and will not be given early.

2. Grading Policy:

   Final grades will be based on an accumulation of up to 500 points, and based on the following scale:

   A	90 - 100%
   B	80 - 89%
   C	70 - 79%
   D	60 - 69%
   F	≤ 59%

Course Requirements

Project Proposal (50 pts): The description of your proposed project should be in a formal written format, and should be prepared using a word processor. You will be turning in a draft proposal and subsequently revising it based in part on the instructor's comments. The proposal should include the following parts:

1. Introduction
   • morphological background information with citations incorporated into the text
   • prioritized list of specific morphological points to be investigated
2. Methods
   • TEM - types of sections needed in order to obtain desired information.
   • SEM - types of surfaces/fractured structures needed in order to obtain desired information.
3. Expected Results and Significance of Project
   • justification for the project - Why is it interesting or important, and how does it relate to existing knowledge?
4. Literature Cited
   • list of references relevant to your particular topic and cited in the proposal. References should include some primary literature, and should focus largely on morphology and electron microscopy.

TEM Proficiency Exam (25 pts):

1. Orally articulate precautions associated with microscope use.
2. Orally articulate start-up procedure associated with microscope use.
3. Know basic parts of the JEOL 100-SX TEM and their functions.
4. Obtain a beam and properly saturate filament.
5. Demonstrate proper specimen exchange (insertion and removal).
6. Demonstrate proper manipulation of grid while in the microscope.
7. Demonstrate proper magnification change, illumination adjustments, and focusing.
8. Demonstrate proper use of camera (expose a micrograph or two).
9. Demonstrate proper method for removing exposed negatives from the TEM.
10. Orally articulate shut-down procedure associated with microscope use.

First Ultrathin Sections (25 pts):

1. Demonstrate proper block size.
2. Demonstrate floating ribbons/sections in a glass knife boat.
3. Demonstrate silver and gold sections.
4. Demonstrate sections without knife marks, chatter, etc.
5. Demonstrate proper collection of sections on copper grids.

TEM negative Series (25 pts):

1. At least 6 developed negatives; three should be of the exact same image and exposed in the TEM using the same illumination parameters. These three should be developed for a different time to reveal the influence of development time on negative contrast.
2. Negatives demonstrating proper illumination and developing procedures.
3. Exposure/development record sheets for the properly illuminated/developed negatives.
4. Written interpretation of the effects of exposure/development time on negative quality (i.e., darkness and contrast).

TEM Print Series (25 pts):

1. At least four high quality prints from four different negatives. Prints should be produced using the Besseler enlarger and should be a minimum of 4 x 5" in size (they can also be larger), and should include the exposure parameters recorded on the back of each print in pencil (e.g., F-stop, exposure time, filter number).
2. The four prints should be accompanied by written comments about the images, including (a) general observations about the subject, (b) conclusions and questions concerning the subject, and (c) a technical critique of each photo.
3. A second set of three prints illustrating a through focus series of the same subject. One negative/print in optimal focus, one underfocused (counter-clockwise from focus), and one overfocused (clockwise from focus); be sure to label each.

Mounted SEM Specimens (25 pts):

1. Orally articulate various methods to mount specimens, and explain why you selected your particular mounting medium.
2. Demonstrate properly mounted specimens (which had previously been critically point dried) on stubs.
3. Demonstrate proper use of the Anatech Hummer sputter coater.

SEM Proficiency Exam (25 pts):

1. Orally articulate precautions associated with microscope use.
2. Orally articulate start-up procedure associated with microscope use.
3. Know basic parts of the JEOL JSM-6100 SEM and their functions.
4. Demonstrate proper specimen exchange (insertion and removal).
5. Obtain a beam and properly saturate filament.
6. Demonstrate proper manipulation of specimen stage while specimens are in the microscope.
7. Demonstrate proper magnification change, probe current adjustments, correction of astigmatism, and focusing.
8. Demonstrate proper method of setting the wave form monitor in order to obtain a high quality micrograph.
9. Demonstrate proper use of camera (expose a micrograph or two).
10. Demonstrate proper method of processing Polaroid PN 55 negatives.
11. Demonstrate proper method of turning off the high voltage.
12. Orally articulate shut-down procedure associated with microscope use.

SEM Print Series (25 pts):

1. At least four high quality prints from four different negatives. Prints should be produced using the Arkay contact printer, and should include the exposure parameters recorded on the back of each print in pencil (e.g., F-stop, exposure time, filter number).
2. The four prints should be accompanied by written comments about the images, including (a) general observations about the subject, (b) conclusions and questions concerning the subject, and (c) a technical critique of each photo.

Written Exam (100 pts):

1. Written exam covering practical and theoretical aspects of TEM and SEM preparation and operation. Exam will consist of multiple question types (objective, short-answer, and long-answer).

Oral Project Report (75 pts): A 15 minute oral presentation describing the results of your research project.

1. Presentation using 35 mm transparencies.
2. Presentation to include an introduction (use "text" slides to provide background and statement of problem), results (interpretations of specimens shown in copy-stand prepared direct positive slides prepared from your TEM and SEM micrograph prints), and discussion/conclusions (using "text" slides).
3. Written critique of your own presentation (note strong points, weak points, specific suggestions for improvement, etc.); due the next day.

Final Written Project Report (150 pts): A written paper based on your project proposal and including the sections listed below.

1. Title (should be informative)
2. Introduction (should include background information and relevant literature citations)
3. Methods and Materials (should include specifics related to your project)
4. Results (should be written in text form and make reference to figures. Figures should include an assortment of well printed, labeled TEM and SEM micrographs and, if appropriate, light micrographs and/or drawings. Each figure should be accompanied by a caption that states the subject of the figure, provides functional interpretation of the morphological data shown in the figure, identifies labels, and indicates the size of the scale bar.)
5. Discussion (optional)
6. Literature cited (list of all references cited in the report)

Course Goals

1. Technical Skills
1. Specimen preparation for transmission electron microscopy (TEM)
   1. Specimen preparation for transmission electron microscopy (TEM)
   • Fixation and dehydration
   • Embedding
   • Ultrathin sectioning (ultramicrotomy)
   • Staining grids with heavy metal stains
   2. Specimen preparation for scanning electron microscopy (SEM)
      • Fixation and dehydration
      • Critical point drying
      • Mounting specimens on stubs
      • Sputter coating
   3. Practical operation of the transmission electron microscope (JEOL 100-SX)
      • Start-up and shut-down
      • Specimen exchange
      • Obtaining and aligning the electron beam
      • Magnification, illumination, and focusing adjustments
      • Exposing micrographs
      • Removing exposed micrographs
      • Processing 3.25 x 4" sheet film
   4. Practical operation of the scanning electron microscope (JEOL JSM-6100)
      • Start-up and shut-down
      • Specimen exchange
      • Obtaining and aligning the electron beam
      • Stage manipulation
      • Magnification, probe current, astigmatism, and focusing adjustments
      • Adjusing the wave form monitor and exposing micrographs
      • Processing 4 x 5" Polaroid Positive/Negative film (PN 55)
   5. Perfection of darkroom techniques
      • Enlarger printing
      • Contact printing
2. Conceptual Aspects
   1. Theoretical knowledge about microscopy (light microscopy [LM], as well as TEM and SEM)
   • Applications of LM, TEM, and SEM
   • Illumination sources (photons vs. accelerated electrons)
   • Lens systems (glass vs. electro-magnetic)
   • Magnification vs. resolution
   • Specimen-electron beam interactions
   • Factors affecting resolution and contrast
   2. Interpretation of electron micrographs (your own and those of others)
      • Recognizing limitations in terms of morphological information
      • Recognizing various types of artifacts
      • Identifying parts of micrographs
      • Drawing morphological conclusions based on electron micrographs
   3. Familiarity with morphological literature
      • Becoming familiar with relevant journals and books
        • For information on specific techniques/protocols
        • For information on TEM and SEM studies about biological topics
      • Prompting you to think about types of biological questions that can be answered by morphological research.
3. Communication Skills
   1. Developing professional speaking skills in general
      • Oral presentation
   2. Developing professional writing skills in general
      • Project proposal
      • Final written report
   3. Communicating morphological information
      • Describing morphological data to an audience with a varied background in your area
      • Oral presentation
      • Final written report