A rastered light pathway is projected onto a specimen by epiluminescence
Reflected or fluoresced light is imaged back through the objective lens
During the mid-to-late 1980Æs commercial microscopes began to appear that made use of the principle of confocal microscopy. Both full-color visible light instruments and laser instruments have been designed and useful in different applications. Both have similar principles of design, however. A beam of light is passed through a small opening and may be deflected in a series of lines that constitute a raster which is projected from above the objective lens of a light microscope, through that lens, and onto the surface of a specimen located in the front focal plane of the lens. The pathway of the light will be at its smallest diameter as determined by the point of light source and its crossover on the surface of the specimen. Since light is coming from above the specimen, it is said to represent epiluminescence.
The object being illuminated may be a section or, more often, a layer (or multiple ones) of the specimen. The crossover light, being in a raster, will be projected one point at a time in rapid sequence. Either reflected or fluoresced light can be captured by the objective lens and be conveyed back up the image tube of the light microscope to a dichroic mirror that projects the radiant image towards a pinhole aperture plate. Any light that was received from either above or below the focal plane of the objective lens will strike the aperture plate away from the pinhole and will be blocked. Only those light paths that have come from the exact focal plane on the specimen will be focused through the pinhole. Such light is then picked up by a photomultiplier tube and used to create a point of some brightness (depending on the amount of light captured through the pinhole) on a cathode-ray tube or computer monitor screen. The position of each point of light displayed will correspond with the position of the rastered laser beam on the specimen. Thus, a scanning laser confocal image can be generated.
