Dr. Jeffrey M. Osborn

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

Advanced Topics: 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

   Microscopy (Biology 518) is primarily a hands-on, research-oriented class. As such, very few to no lectures will be presented. Specific readings, however, will be assigned, and students will be expected to demonstrate comprehension of the assigned material via incorporation into their lab work.

   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:

   Grades will be based on an accumulation of up to 600 points.

   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
   • LM - types of sections needed to obtain desired information
   • SEM - types of surfaces/fractured structures needed to obtain desired information
3. Expected Results and Significance of Project
   • 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 microscopy.

Embedded Specimens (25 pts):

1. Your project specimens, fixed and embedded in low viscosity epoxy resin.
2. Fixation and embedding records for your embedded specimens.

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 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 for digital acquisition and printing images (use of the Codontics printer).
12. Demonstrate proper method of turning off the high voltage.
13. Orally articulate shut-down procedure associated with microscope use.

SEM Micrograph Series (Digital Images) (25 pts):

1. At least four high quality prints from four different negatives. Prints should be produced using the dye sublimation printer, and should include the image parameters recorded for each print.
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 micrograph.

Ultramicrotome Proficiency Exam (25 pts):

1. Demonstrate operation of glass knife maker.
2. Orally articulate precautions associated with ultramicrotome use.
3. Know parts of the ultramicrotome and their functions.
4. Demonstrate microtome operation using (a) a specimen that you have properly trimmed and mounted, and (b) a good glass knife that you have prepared.

First Stained Sections (25 pts):

1. One labeled microscope slide showing specimen properly cut and stained, and with coverslip properly mounted.
2. Oral interpretation of specimen on slide.

Light Microscopy Proficiency Exam (25 pts):

1. Orally articulate precautions associated with microscope use.
2. Know parts of the Olympus photomicroscope and their functions.
3. Adjust field diaphragm, substage condenser and substage diaphragm to obtain Koehler illumination.
4. Demonstrate method of increasing contrast for transparent specimens.
5. Demonstrate method of changing objectives.
6. Demonstrate proper adjustment of Olympus photomicroscope to yield a good darkfield image.
7. Demonstrate proper adjustment of Olympus photomicroscope to yield a good Nomarski-DIC (differential interference contrast) image at all possible magnifications.
8. Demonstrate method of using the Sony video camera and digital image acquisition and archiving.

Light Microscopy Proficiency Exam (25 pts):

1. At least four high quality prints from four different images. Prints should be produced using the dye sublimation printer, and should include the image parameters recorded for each print.
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 micrograph.

Oral Project Report (100 pts): A 15 minute oral presentation describing the results of your research project.

1. Presentation using Microsoft PowerPoint.
2. Presentation to include an introduction (use "text" slides to provide background and statement of problem), results (presentation and interpretations of a] thick-sectioned specimens imaged with the LM and of 2] whole and sectioned specimens imaged with the SEM), 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 LM and SEM micrographs. 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 (should be synthetic, by placing your results in a broader context - i.e., interpretation of your results given the what information exists in the literature base).
6. Literature cited (list of all references cited in the report)

Course Goals

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. Technical Skills
   1. Specimen preparation for transmitted light microscopy (LM)
   • Fixation and dehydration
   • Embedding
   • Thick sectioning (ultramicrotomy)
   • Staining sections with chromatic 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 research-grade compound light microscope (Olympus BHS)
      • Obtaining Koehler illumination
      • Objective changes
      • Use of various contrast methods (brightfield, darkfied, and differential interference contrast)
      • Cleaning glass surfaces
      • Use of the 35mm camera system
   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. Digital image techniques
      • Image acquisition
      • Image manipulation
      • Image output
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
   2. Interpretation of 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 micrographs
   3. Familiarity with morphological literature
      • Becoming familiar with relevant journals and books
        • For information on specific techniques/protocols
        • For information on morphological 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