Three-Dimensional Confocal Light Microscopy of Neurons: Fluorescent and Reflection Stains

Confocal imaging is highly dependent on contrast enhancement methods to increase the visibility of the details of interest relative to their surroundings. This is particularly important for neurons embedded in thick tissue slices because they are surrounded by large numbers of other cells with identical optical properties. Thus staining and specimen preparation procedures are of critical importance, and it is essential to use selective stains that label only the portion of the specimen that is of interest. This specificity can be achieved by filling a selected space with stain, by exposing the specimen to stains that chemically bind to a particular component(s), or by conjugating the stain to a lectin or antibody that binds specifically to a particular component. There are two general categories of stains used for confocal imaging applied to neurobiology: fluorescent and reflection. When using the fluorescent, the illuminating light excites a molecular transition that decays by emitting light at a wavelength longer than the incident light, and the optical system allows only the longer wavelength to be detected. The latter reflects a portion of the incident light back into the objective lens to be subsequently detected to form the image.