Principles of Fluorescence [5]

Spectroscopy Luminescence is the emission of light from any substance, and occurs from electronically excited states. Luminescence is formally divided into two categories, -fluorescence and phosphorescence-depending, on the nature of excited state. In excited singlet state, the electron in the excited orbital is paired (by opposite spin) to the second electron in the ground-state orbital. Consequently, return to the ground-state is spin allowed and occurs rapidly by emission oh a photon. Phosphorescence is emission of light triplet excited states, in which the electron in the excited orbital has the same spin orientation as the ground-state electron. Transition to the ground- state are forbidden and the emission rates are slow. Fluorescence spectral data are generally presented as emission spectra. A fluorescence emission spectrum is a plot of the fluorescence intensity versus wavelenght (nm) or wavenumber (cm-1 ). Emission spectra vary widely and are dependent upon the chemical structure of the fluorophore and the solvent in which it is dissolved. An important feature of fluorescence is high sensitivity detection. The processes that occur between the absorption and emission of light are usually illustrated by the Jablonski diagram . A typical Jablonski diagram is shown in Figure 1. The singlet ground, first, second electronic state are depicted by S 0 , S 1 and S 2 respectively. At each of these electronic energy levels the fluorophores can exist in a number of vibrational energy levels, decicted by 0, 1 , 2 etc. The transition between states are depicted as vertical lines to illustrate the instantaneous nature of light absorption. The larger energy difference between the S 0 and S 1 excited states is too large for thermal population of S 1 . For this reason you use light and not heat to induce fluorescence. Following light absorption, several processes usually excited to some higher vibrational level of either S 1 or S 2 . Molecules in condensed phase rapidly relax to the lowest vibrational level of S 1 . This process is called internal conversion and occurs within 10 -12 s or less. Since fluorescence lifetime are typically near 10 -8 s, internal conversion is generally complete prior to emission. Hence, fluorescence emission generally results from a thermally equilibrated state, that is, the lowest energy vibrational state of S 1.

FIG 1: Jablonski Energy Diagram



The bioluminescence of the primary photoprotein is blue. The bioluminescence from the jellyfish is green due to a closely associated green fluorescent protein. GFP contains a highly fluorescent group within a highly constrained and protected region of the protein. The chromophore is contained within a barrel of beta - sheet protein in Fig 2.

FIG 2: the cromophore

Tsien R., Annual Review of Biochemistry, 1998, 67, 509-544

The remarkable feature of the GFP is that the chromophore forms spontaneously upon folding of the polypeptide chain without the need for enzymatic synthesis in Figure 2. As a result, it is possible to express the gene for GFP into cells, and to obtain proteins which are synthesized with attached GFP. It is even possible to express GFP in entire organism. In general GFP have good photostability and display high quantum yields, which is probably because the b- barrel structure shields the chromophore from the local environment.

For a time it was thought that GFP was only present in Aequorea Victoria. It is know that similar naturally fluorescent proteins are present in a number of Anthrozoa species, in coral. As a result, a large number of fluorescent proteins are now available with emission maxima ranging from 448 to 600 nm.