RRC-CMF: Microscopy Tutorials

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Fluorescent and transmitted light microscopy techniques are constantly evolving. Yet as new applications and innovations are developed for use with cells or tissues, many of the same core microscopy principles remain essential. The links below expand on these principles, and have been compiled to include some of the best tutorials, guides, and protocols available.

For details and downloadable manuals related to a specific microscope, please visit the corresponding instrument page.

Contents

Basic Microscopy

•  Introduction to Microscopy
A primer on the fundamental concepts behind magnification (from Zeiss)

•  Image Formation in the Microscope
An easy-to-follow review of the underlying physics of all microscopes (from Zeiss)

•  Numerical Aperture and Resolution
A summary of how key properites of a microscope objective can influence the characteristics of an image (from Zeiss)

•  Illumination and the Optical Train
A straightforward discussion of key microscope components and the ways in which they are integrated (from Zeiss)

Confocal Microscopy

•  Introduction to Confocal Microscopy
A comprehensive overview of the history and methods involved in optical sectioning (from Olympus)

•  Imaging Modes in Confocal Microscopy
A detailed list of the various modes commonly available on most confocal microscope systems (from Nikon)

•  Comparing Confocal and Widefield Fluorescence Microscopy
An interactive applet that demonstates the qualities of confocal imaging (from Olympus, requires Java)

•  Specimen Preparation and Imaging
A review of certain considerations and expectations important in confocal image acquisition (from Nikon)

•  Critical Aspects of Confocal Microscopy
A listing of the various tradeoffs between the numerous optical and mechanical components within a confocal microscope that can directly affect image quality (from Nikon)

Fluorescent Probes

•  Excitation of Fluorescent Probes
A brief description of the nuance behind fluorescent probe excitation efficiencies, which must often be considered when designing experiments around available filter sets (from Olympus, requires Java)

•  Spectral Crosstalk in Microscopy
An excellent summary on how to avoid or correct "bleed-through" of fluorescent signals (from Olympus)

•  FRET Biosensors
An interactive tutorial showing an example of the resonant energy transfer process (from Nikon, requires Flash)

Image Analysis

•  What's in a Picture? The Temptation of Image Manipulation
A brief refresher on the do's and don'ts of fine-tuning fluorescent microscope images (from Journal of Cell Biology)

•  How to Quantify Colocalization in Images
A detailed guide demonstrating the colocalization measurement tools available in Zeiss confocal microscope software (from Optical Biology Core, University of California)

•  Understanding Colocalization Coefficients
A step-by-step walkthrough of how the most popular ways of quantifying colocalization are calculated (from Scientific Volume Imaging")

•  FRET Measurement Setup for Donor-Acceptor Pairs
A useful protocol that describes the most common approach for acquiring and analyzing FRET readouts (from Lamond Lab, University of Dundee)

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