In Laser Scanning Confocal Microscopy, a laser beam is scanned across the specimen and the emitted fluorescent light passes through a pinhole aperture, so that the fluorescence from points on the specimen that are not within the focal plane will be obstructed by the pinhole. This nearly eliminates out-of-focus information, improving resolution and background discrimination. This 'optical sectioning' is especially useful when viewing thick specimens. A series of sections can be obtained automatically and a 2-D or 3-D reconstruction created. Signals from 3 different fluorescent dyes or 2 dyes and a transmitted light image can be obtained simultaneously and the images merged to determine co-localization.
In Multiphoton Microscopy, a more powerful laser is used to provide simultaneous excitation with 2 or three photons of low energy, exciting the fluorophore to the same level as one high energy photon. The use of lower energy (red wavelengths) reduces damage to the specimen, which is especially important in living tissue. These wavelengths also penetrate deeper into the tissue. Multiphoton excitation occurs only at the focal plane and tissue above and below is not affected so there is no need for pinhole apertures. In confocal a larger volume if tissue is excited but only light from the focal plane is collected.