Fluorescence is a molecular phenomenon in which a substance absorbs light (excitation) and radiates light of longer wavelength (emission) for a very short period of time. When a fluorochrome absorbs light, energy in the form of photons is taken up, leading to excitation of electrons to higher energy states. The process of absorption of photons is extremely rapid and is immediately followed by a return to lower energy states, which is then accompanied by emission of photons, which can be observed if light is emitted in the visual range.
This short duration (nanoseconds) distinguishes it from other forms of luminescence such as phosphorescence. The difference in wavelengths between the excitation and emission peaks is referred to as the Stokes shift. Fluorochromes with a large Stokes shift are easy to excite and to observe their emission in a fluorescence microscope. With transmitted light illumination, a small Stokes shift may make it impossible to illuminate a fluorochrome at its excitation peak and to observe the fluorescence colour at its emission peak.
Confocal laser scanning fluorescence microscopy enables two-photon excitation with photons of longer wave-length than the emitted light. The electron is then brought into its excited state with two photons of half the required energy, arriving simultaneously. In most cases the electron ends up in the same excited state as with normal single-photon excitation before it drops down to the ground state. Similarly, the fluorescence emitted is similar to that given off by normal single-photon excitation.
Many molecules, both non-organic and organic, show fluorescence emission, especially with excitation using high energy radiation. When plant or animal tissue is excited with UV-light very often a bluish fluorescence emission is observed which is called primary fluorescence, or autofluorescence [7, 8]. Naturally occurring substances often have very broad excitation and emission spectra. With the development of molecular biology, research has been focussed on special dyes with bright fluorescence to distinguish them from possible autofluorescence. The dyes used to selectively stain biologically important molecules are called fluorochromes. When conjugated to antibodies or nucleic acids, they are referred to as fluorescent markers or probes. Today fluorochromes are available with peak emissions in the violet, blue, green, orange, red and near-infrared regions of the spectrum. The possibility to excite fluorochromes with orange and red light and detect the red and infrared fluorescence with a photomultiplier or a