Fluroscence:

Fluorescence is the process distinct from incandescence .Heating is generally detrimental to the process of the fluorescence, and most substances, when fluorescing, produce little heat. For this reason fluorescence is commonly referred to as "cold light".

In the standard conceptual model of molecules electrons occupy distinct orbits and therefore energy levels. This is a simple view of molecules, but it serve to outline the process of fluorescence. A common result of directing light on a molecule which absorbs rather than transmits is that one or more of the electrons of the molecule are "kicked" into a higher energy state. All these excited electronic states are unstable and later or sooner the electrons will lose their excess energy and fall back to lower energy states. This excess energy can be dissipated in a number of ways, the most common being simply to increase atomic vibrations within the molecule. But some of the molecules are capable of emitting some of the energy as light. This is the phenomenon we see as fluorescence. Fluorescence decay of the pure sample showing the single exponential decay. The dark line shows excitation pulse. Time correlated single photon counting was used to obtain the data. This technique counts the several emitted photons hitting a detector at times, t, following excitation.

One critical difference between steady-state and kinetic measurements of the fluorescence is that the value of t_F is not a function of concentration of sample while the value of F_F is concentration dependent. At low concentration is the value of F_Flinearly dependent on the concentration. The reason is inner filter effect. Fluorescence emission spectra of human serum albumin, tryptophan alone, and an 18:1 molar ratio of tyrosine and tryptophan: Excitation at 245 nm.

The 18:1 sample approximates relative occurrence of these amino acids in protein. The spectrum of the protein closely resembles that of the pure tryptophan because tyrosine sensitivity is low and its emission is most probable quenched by tryptophan.