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Introduction to Confocal Microscopy & Image Analysis

BMS 524, 2 Credit Course Lectures: Spring 1997, Practicals Maymester 1997

Professor: J.Paul Robinson
Ideal for Graduate Students, Seniors - Faculty & Staff invited to monitor

Course Description

Confocal Microscopy provides a powerful tool for evaluation of the 3D structure of a variety of tissues and biological samples, as well as some non-biological samples. In addition, using the multiple excitation capacity of these instruments it is possible to identify multiple sites of interest within a single sample and to reproduce these data using image reconstruction techniques. Live cells can also be evaluated using these technologies to monitor calcium fluxes, pH changes or production of oxygen and nitrogen radicals using fluorescent probes.

Participants will become familiar with the rudiments of confocal and fluorescent microcsopes, and associated technologies. They will learn the fundamentals of fluorescence detection and develop a keen understanding of the use of and availability of a variety of fluorescent probes across the UV and visible spectrum. They will also be introduced to basic image analysis techniques.

Participants will learn to use both the UV/visible confocal scope and the associated operating software. They will have the opportunity to run the instrument in small groups and will be required to produce images demonstrating their capability. They will also learn the use of the real-time linescan confocal scope using video acquisition software including Universal Imaging Metamorph software.

At the end of the course, all participants should be capable of understanding the theory and should be capable of essentially operating instrumentation currently housed in the Cytometry Laboratories at Purdue University.

The course is STRICTLY LIMITED because of the need for practical participation. This is the first such course offered at Purdue University. Acceptance into the course will be at the instructor’s discretion; however, current graduate students will be given priority. There are NO PREREQUISITES.

Course Structure:

Instructor: J. Paul Robinson
Lectures: 16 hours (Spring semester)
Practical Demonstrations: 10 hours (Maymester)
Practical Participation: 20 hours (Maymester)
Duration: January 13-June, 1996
Tentative Schedule (may change): Lectures will be Wednesdays 11:30 - 12:20 during Spring semester. Practicals will be given during Maymester: practical demonstrations will be Tuesday 1-3 pm; Thursday 1-3pm; practical (hands-on) sessions will be Tuesday and Thursday 3-5 pm.

This course is offered through the Purdue University Cytometry Laboratories which is under the Department of Basic Medical Sciences in the School of Veterinary Medicine. Funding for the Course is provided by the Department, the Vet School, and via the Vice President's Reinvestment Fund.


(Given by Dr. J. Paul Robinson unless otherwise indicated)
1. The Principles of Fluorescence Microscopy
2. The Principles of Confocal Microscopy
3. Fluorescent Probes
4. Sample Preparation for Confocal Microscopy
5. Image Formats and Image Manipulations
6. Introduction to the Bio-Rad MRC 1024 Confocal System
7. Introduction to the Bio-Rad DVC Viewscan System
8. Principles of 2D image analysis - Morphometry(I) (Dr. John Turek)
9. Applications of Morphometry (II) (Dr. John Turek)
10. 3D Reconstruction
11. Confocal Microscopy for Neuroscience (Dr. Terry Powley)
12. Live Cell Applications
13. Calcium Imaging Applications (Dr. Ken Robinson)
14. Applications in Immunology (TBA)
15. Advanced Applications of Imaging: 2 Photon Imaging (TBA)
16. Course Overview

Grading: Final Examination: 65% Practicals - preparation & analysis of appropriate images: 35%
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For information contact J.Paul Robinson, Ph.D., Director PUCL, 1515 Hansen B050, Purdue University, West Lafayette, IN 47907-1515, U.S.A. Phone: (317) 494-0757; Fax: (317) 494-0517;