Biology 725: Microscopy and Image Analysis

Biology 725: Microscopy and Image Analysis. Concepts and theory involved in a wide variety of microscopy and image analysis.  The course is intended for people who will do a significant amount of biological imaging in their graduate research and is best taken after starting to do some practical microscopy.

Areas covered include fluorescence, components and principles of imaging modalities: transmitted light microscopy, widefield imaging, point-scanning confocal, TIRF, live-cell imaging, multiphoton, spinning disk and super-resolution microscopy, imaging techniques, image processing, visualization and analysis. FIJI/ImageJ is used with interactive exercises to complement theory involved in image processing and analysis. Consent of the instructor is required (numbers are limited). Instructor: Sam Johnson. 2 credits.  

Sam Johnson
4215 FFSC


Prerequisite: Some experience with practical microscopy, even if quite basic, is important to get the most out of the course. You certainly don't need to have used all the imaging modalities covered in the course but if you have never touched a microscope before this course might not make sense.

Enrollment: If you are interested in taking this course please request enrollment with this form with some details of what you are doing and why you want to do the course. Since the numbers are limited, places are given to those who would seem to benefit most from the course. Official enrollment is through ACES with a permission number.

Outline of semester by week

The course is mainly theory-based as it is designed to complement regular use of microscopes in your research. Sessions that are mainly practical are marked below in orange and will involve small groups using the equipment. The times for the practical work may be slightly different to the normal course hours.

General theme
Topics covered
Core Microscopy

Introduction to the course • Fluorescence • Fluorophores and fluorescent proteins • Samples • Optical principles • Components of a microscope • Fluorescence filters and dichroics • Optimal objective choice • Cameras
Transmitted light • Kohler illumination • Brightfield, Darkfield, Phase, Pol and DIC Kohler illumination • Setting up phase contrast, DIC • Questions about everything so far
Optical sectioning

Introduction to point scanning confocals
Advanced confocal • Multi-photon • Spinning disk • TIRF • SPIM • Deconvolution •
9 Computer practical Image processing and visualization

Super resolution microscopy

STED • Structured illumination • PALM/STORM • EM
Live cell imaging • Photokinetics • FRET • Photocontrol
Images analysis

Quantification and analysis principles • Interactive examples with FIJI/ImageJ (bring your computer with FIJI and the example files installed)

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