CMB551 module 1A: Microscopy and Image Analysis in Cell Biology

Summary: Microscopy has been revolutionized by fluorescence and now provides a vast array of tools with which to investigate biology. This module will cover the principles and possibilities of microscopy – how microscopes and photon‐based imaging systems work and what you can do with them. How do you visualize the morphology of microscopic objects using light and fluorescence? Which imaging modality is best for a particular sample? How do you gain information on the dynamics of systems such as the spatial and temporal patterns of signaling events? How do you extract quantitative information from images? We will discuss a range of techniques with a heavy emphasis on imaging living samples from microbes to vertebrate animals ‐ widefield imaging, optical sectioning by confocals, multi‐ photon excitation, TIRF, protein dynamics, choosing and exploiting fluorescent proteins/probes and super‐resolution microscopy. The theory and physical principles of the imaging systems will be explained in the first half of the module to a level giving understanding of how they work and guidance for optimal use. The second part of the module will be a mixture of theory and exercises in FIJI/ImageJ covering the processing, visualization and quantification of microscopy data.

Readings: from "The Molecular Biology of the Cell", Alberts et al. Chapter 9 – focus on the sections discussing light/fluorescence microscopy

Instructor:
Benjamin Carlson
benjamin.carlson@duke.edu
681-4076
337 Nanaline Duke
31 August - 12 September 2016
MWF 10:20-11:40
384 Nanaline Duke

Course outline and content

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3

Principles of microscopy - Fluorescence, core concepts, objectives, cameras, filters, transmitted light and contrast. Optical sectioning: confocals and 3D imaging - The confocal principle, lasers, scanning, SNR improvements, detectors, sampling, point-spread function. limitations of point scanning confocals, spinning disks, TIRF, multiphoton, SPIM. Review and comparison. Super resolution overview - three families of techniques that improve the resolution beyond the diffraction limit.

Slides

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Day 2

Day 3

H/W

Techniques - Immunofluorescence, fluorophores, live cell imaging, fluorescent proteins, photokinetics, protein-protein interactions, photocontrol, reporters, miscellaneous.

Material to read through on your own

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Images and analysis - Principles and examples using FIJI/ImageJ: image formats, histogram, scaling and digital contrast, display and visualization. Bring your computer and follow along with the exercises.

Slides

Day 1

Day 2

Day 3


FIJI install | Dataset

 

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