CMM5360 Current Topics in Biological Imaging (3 cr.)

Modern methods in optical microscopy have provided revolutionary improvements in our ability to view molecular mechanisms in cells.  This course touches on all aspects of biological imaging, providing a systematic and in-depth examination of the theory of image formation and the application of diverse imaging techniques to explore biological questions at the level of the single cell.  In particular, the course emphasizes the importance of proper sample preparation and highlights quantitative issues that are critical to the proper interpretation of images obtained with modern microscope systems.  Students will also learn how to critically read primary literature based on imaging approaches. 

This course will be offered in Winter 2012 (Mondays, 2-5 p.m.).

Students can pre-register by sending an email (which includes your name, program, supervisor and a brief description of how you are using microscopy in your research project) to Dr. Laura Trinkle-Mulcahy (ltrinkle@uottawa.ca), and will then be informed if space is available.

Course Format

Each 3 hour class will consist of 1.5 hours lecture and 1.5 hours assigned literature discussions.  The lecture will cover the background and key concepts that are needed to critically evaluate the assigned reading/literature presentations.  For each lecture, one or two students (depending on class size) will be assigned current papers related to the topic, and asked to give a short presentation (15-20 min) on how a particular imaging method contributed to the results and impact of the research.  All students in the class will be expected to read these papers and to participate in the discussion.

Students will also prepare a manuscript review in the format of a formal review of a submitted manuscript, as if the student were reviewing it prior to acceptance for publication.  The manuscript will be assigned by the course co-ordinator and Journal of Cell Biology guidelines provided.  Students will be assessed on writing clarity, scientific accuracy and relevance.

As a final project, students will prepare a comprehensive review of an advanced imaging technique.  Approval for the topic must be obtained from the course coordinator.  During the last 2 sessions each student will give a short (10-15 min) presentation in which they briefly outline the design of an experiment, using their chosen technique, that they would use to probe a question in their own research project. 

Evaluation

Participation (attendance and participation in class discussions; evaluated by LTM) 10%
Manuscript review following JCB guidelines 10%
Seminar (15-20 min) presenting a paper assigned by the lecturer 30%
Review of advanced imaging technique and short presentation (10-15 min) 50%

Lecturers

Drs. Laura Trinkle-Mulcahy ( Course Coordinator), Jean-Claude Béïque and William Staine..  Two guest lectures will be given by visiting professors (TBA).

Brief Outline of Topics that will be Covered

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 Introduction: full description of course including grading scheme. History of microscopy and review of the basics (magnification, resolution and contrast; principals of image formation; aberration and diffraction).
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 Principles of fluorescence and fluorescence microscope designs: will include comparisons of widefield fluorescence and confocal laser scanning microscopy systems and introduce specialized systems such as TIRF, structured illumination and STED (and PALM/STORM)
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 Fluorophores: dyes, intracellular markers and genetically encoded fluorescent proteins, including a discussion of the unique properties of GFP variants and their use in advanced imaging techniques; excitation and emission spectra
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 Sample preparation and immunological methods: optimizing fixation and immunological staining in cells and tissue sections; discussion of multiwavelength imaging, fluorophore bleedthrough and quantification of fluorescent signals.
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 Electron microscopy: Basics of TEM and SEM, including sample preparation and data collection and analysis.
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 Confocal and multiphoton microscopy: principles of confocal and multiphoton microscopy and their application cell imaging, in vivo and deep tissue imaging, calcium imaging, photo-uncaging, 2nd harmonic generation and fluorescence lifetime imaging (FLIM)
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 Image acquistion, processing and feature extraction: discussion of digital imaging and detector systems, highlighting quantitative issues and discussing ethics of digital data manipulation
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 Live cell imaging: will include discussions of resolution vs. signal intensity, generation of 3D multiwavelength time lapse movies, and nondestructive techniques such as phase contrast polarization and differential interference contrast.
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 Advanced imaging techniques - colocalization and direct protein-protein interaction: will discuss markers for intracellular structures and principles and the application of fluorescence resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC) techniques to measure direct protein-protein interaction in vivo.
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 Advanced imaging techniques – photobleaching, photoactivation and photoconversion: will discuss the application of photokinetic imaging methods to measure dynamic properties of proteins in live cells. 
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 Diagnostic Imaging: advanced topics in diagnostic imaging techniques.
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 2-3 sessions of student presentations (dependent on number of students); 10-15 min talk followed by discussion.











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