Electrons in this state are unstable and will return to the ground state, releasing energy in the form of light and heat. This emission of energy is fluorescence. The dye glows brightly against a dark background because only the emitted wavelength is allowed to reach the eyepieces or camera port of the microscope. Most microscopes are designed using epi-illumination. In epi-illumination excitation, light goes through the objective lens and illuminates the object.
Light emitted from the specimen is collected by the same objective lens.
Sometimes the fluorescent molecule itself is a direct stain or probe for specific structures. In other situations the fluorescent dye is bound to another non-fluorescent probe that recognizes specific structures. For example, the fluorescence molecule, rhodamine may be conjugated to phalloidin, which binds the filamentous actin. One important method to identify specific proteins is to couple fluorescent dyes to antibodies that bind very specifically to macromolecules in the cell. Sometimes the fluorescent molecule itself is a direct stain or probe for specific structures.