After the antibodies are isolated and purified, a fluorescent dye, fluorescein, becomes attached to them by a chemical reaction. The fluorescence microscope reveals the sites containing the antigen-antibody complex as bright luminescent areas in a dark background. The point is a diffraction limited spot on the specimen and is produced either by imaging an illuminated aperture situated in a conjugate focal plane to the specimen or, more usually, by focusing a parallel laser beam. With only a single point illuminated, the illumination intensity rapidly falls off above and below the plane of focus as the beam converges and diverges, thus reducing excitation of fluorescence for interfering objects situated out of the focal plane being examined. Any light emanating from regions away from the vicinity of the illuminated point will be blocked by the aperture, thus providing yet further attenuation of out-of focus interference.
Light passing through the image pinhole is detected by a photodetector.
Usually a computer is used to control the sequential scanning of the sample and to assemble the image for display onto a video monitor. Most confocal microscopes are implemented as imaging systems that couple to a conventional microscope. The resolution obtained by a confocal microscope can be better by a factor of up to 1.4 than the resolution obtained with the microscope operated conventionally. This application is often used to image the surface of specimens. The Internet Atlas of Histology .