UNIT 2.2Basic Confocal Microscopy

Confocal microscopy produces sharp im-ages of structures within relatively thick speci-mens (up to several hundred microns). It is particularly useful for examining fluorescent specimens. Thick fluorescent specimens viewed with a conventional widefield fluores-cent microscope appear blurry and lack contrast because fluorophores throughout the entire depth of the specimen are illuminated and fluo-rescence signals are collected not only from the plane of focus but also from areas above and below. Confocal microscopes selectively col-lect light from thin (∼1-µm) optical sections representing single focal planes within the specimen. Structures within the focal plane appear more sharply defined than they would with a conventional microscope because there is essentially no flare of light from out-of-focus areas. A three-dimensional view of the speci-men can be reconstructed from a series of optical sections at different depths. The confocal microscope is the instrument of choice for examining fluorescence-stained neurons in brain slices (Fig. 2.2.1) or small, intact organisms such as Drosophila and ze-brafish embryos. It is also useful for localizing intracellular antigens in dissociated cells. Its sensitivity even allows fluorescence in living specimens to be monitored, making it feasible to follow the movements in living cells of fluorescent probes such as green fluorescent protein (GFP; Fig. 2.2.2), dyes for specific organelles, FM-143 (a marker for synaptic ves-icle recycling), DiI (a dicarbocyanine dye that stains the plasma membrane), and Ca2+ indica-tors (e.g., Fluo-3, calcium green, and Indo-1; see UNIT 2.3). In addition, some types of confocal microscopes can be configured to perform pho-tobleach experiments (Fig. 2.2.2C, D) and to photoactivate “caged ” molecules (molecules that are inactive until released with UV illumi-nation). Neurobiologists use confocal microscopy in a number of creative ways that are beyond the scope of this article. The information presented herein is intended to provide background and practical tips needed to get started with confo-cal microscopy. An excellent source of theoreti-cal and technical information is the Handboo