Why Use a Confocal Microscope?
The most important feature of a confocal microscope is the capability 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.
This figure depicts the effect of the pinhole, or iris diaphragm, on the thickness of the optical plane that is collected. The pinhole and focal plane in the sample are at conjugate planes of focus.
The small pinhole opening in the diagram on the left enables data collection from a thin optical plane within the specimen. Points that are out of the plane of focus (red) will have a different secondary focal plane thus, most of the data is deflected.
Although some of the out-of-focus light enters the photomultiplier tube (PMT) in the figure on the left, the intensity is too dim to be visualized. All of the data at the plane of focus is collected (blue). In this manner, the confocal microscope can collect only the data from within the focal plane. The larger pinhole opening in the figure on the right allows both in-focus and out-of-focus data to be collected.
More information on how a confocal works:
Laser Scanning Confocal Microscope Simulator: interactive tutorial