E-Book Overview
Stephen Paddock and a highly skilled panel of experts lead the researcher using confocal techniques from the bench top, through the imaging process, to the journal page. They concisely describe all the key stages of confocal imaging-from tissue sampling methods, through the staining process, to the manipulation, presentation, and publication of the realized image. Written in a user-friendly, nontechnical style, the methods specifically cover most of the commonly used model organisms: worms, sea urchins, flies, plants, yeast, frogs, and zebrafish. The powerful hands-on methods collected here will help even the novice to produce first-class cover-quality confocal images.
E-Book Content
Methods in Molecular Biology
TM
VOLUME 122
Confocal Microscopy Methods and Protocols Edited by
Stephen W. Paddock
HUMANA PRESS
An Introduction to Confocal Imaging
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1 An Introduction to Confocal Imaging Stephen W. Paddock 1. Introduction The major application of confocal microscopy in the biomedical sciences is for imaging either fixed or living tissues that have usually been labeled with one or more fluorescent probes. When these samples are imaged using a conventional light microscope, the fluorescence in the specimen away from the region of interest interferes with resolution of structures in focus, especially for those specimens that are thicker than approx. 2 µm (Fig. 1). The confocal approach provides a slight increase in both lateral and axial resolution, although it is the ability of the instrument to eliminate the "out-of-focus" flare from thick fluorescently labeled specimens that has caused the explosion in its popularity in recent years. Most modern confocal microscopes are now relatively easy to operate and have become integral parts of many multiuser imaging facilities. Because the resolution achieved by the l