The most important feature of a confocal microscope is the capablity of isolating and collecting a plane of focus from within a sample, thus eliminating the out of focus "haze" normally seen with a fluorescent sample. Fine detail is often obscured by the haze and cannot be detected in a non-confocal, fluorescent microscope.
The
confocal microscope has a stepper motor attached to the fine focus, enabling the collection of a series of images through a three
dimensional object. These images can then be used for a two or three dimensional reconstruction.
Double and triple labels can be collected
with a confocal microscope. Since these images are collected from an optical plane within the sample, precise colocalizations can
be performed.
Protocol 1. Confocal microscope technique ( http://depts.washington.edu/keck/intro.htm)
Protocol 2. Immunofluorescence / confocal microscopy