St. Lawrence University Microscopy & Imagery Center

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Research Methods in Laser Scanning Confocal Microscopy

Bio. 392 (0.5 units)

Dr. T. Budd

Course description: Confocal microscopy allows a fluorescence-labeled specimen to be optically sectioned at magnifications up to 1000x. It is a good bridge between standard fluorescence microscopy and electron microscopy. This course will cover the theory and practice of Confocal Microscopy. Students will learn specimen preparation techniques, fluorescent dye selection, laser physics, optics, as well as digital image acquisition and analysis. As a half unit offering, the course will meet for about half the semester. Meeting times are by arrangement. Prerequisites are any 200 or 300 level science course or permission of the instructor.

Topics for understanding:

Light energy
Fluorescence
Optical systems
Confocal optical systems
Laser light sources
Laser safety
The inverted confocal microscope
Setting the beam source(s)
Resolution
Photomultiplier tubes (PMTs)
Parameter settings in preview mode
Saving a customized template
Single scan of an image
Optical sectioning
Saving and manipulation of image data
Viewing images in LCS
3D imagery
Annotation of images
Printing images

Students taking the confocal course without the project module will be assigned a specimen and "stain" it for resolution of basic cellular structures. These results will be prepared as a lab report and presented orally to their classmates. Students taking the concurrent project module will do a microscopy project of their own design based on primary literature findings and prepare the results as a journal article and orally present the results to their classmates.

Grading will be based on a series of quizzes and practical exams using the confocal microscope. The write up of the sample workup or project will also be graded for quality and quantity of images acquired and on the level of integration of findings with the primary literature.

The grade scale is 90% and above = 4.0, 85-90% = 3.5, 80-85%= 3.0, etc.

There is no textbook for this course. Handouts, lectures and practical demonstrations will be extensively used. It is imperative that students be well rested and alert when using the confocal microscope. Since this course demands manual dexterity and error free precision in thinking, learning and lab skills, students with learning or physical disabilities should self-identify to the instructor before attempting this course so as to determine the probability of success. This course requires extended periods of concentration and considerable time commitment outside of class meeting times. Poor eyesight, fatigue, and poor hand-eye coordination is also a hindrance in mastering these skills. The instrumentation used in this course is valued over $280,000. There is no room for error in its use. Maximum attention to detail and perfection is required.


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