Time-Resolved Fluorescence Spectroscopy with LabView

1.1 Fluorescence phenomenon The absorption and subsequent emission of light by organic and inorganic specimens is typically the result of physical phenomena known as luminescence, which occurs at electronically excited states. Luminescence is formally divided into two categories: fluorescence and phosphorescence, depending on the nature of the excited state. Fluorescence occurs when a photon excites an electron from the ground-state to a higher energy state, and the electron in the excited orbital is paired (of opposite spin) to the second electron in the ground-state orbital. Return to the ground state is spin allowed and occurs rapidly by emission of a photon producing fluorescence. The emission rate of fluorescence (fluorescence lifetime) is of the order of nanoseconds to microseconds. The lifetime ┬ of a fluorophore (fluorescence substance) is the average time between its excitation and its return to the ground state. Phosphorescence is the light emission from excited states, in which the electron in the excited orbital has the same spin orientation as the ground-state electron. Transitions to the ground state are forbidden and the emission rates are slow, in the order of millisecond to seconds (Lakowicz, 1991), (Lakowicz, 1999), (Gore, 2000), (Valeur, 2002). The processes which occur between the absorption and emission of light are usually illustrated by a Jablonski diagram (figure 1). S0, S1 and S2 denote ground, first, and second electronic states. At each of these electronic energy levels the electrons can exist in a number of vibration energy levels denoted by 0, 1, 2, etc. The transitions between states are depicted as vertical lines to illustrate the instantaneous nature of light absorption (10-15s). An electron is usually excited to some higher vibration level of either S1 or S2 and rapidly relaxes (10-12 s–10-15 s) to the lowest vibration level of S1. Return from level S1 to the ground state S0 produces fluorescence emission. Examination of the Jablonski diagram revels that the energy of emission is less than that of absorption. Hence, fluorescence occurs at lower energies or longer wavelength (Stokes shift). The amount of Stokes shift is a measure of the relaxation process occurring in the excited state, populated by absorption. Another property of fluorescence is that the same fluorescence emission spectrum is generally observed irrespective of the excitation wavelength. This is known as Kasha ́s rule. Although fluorescence measurements are more sophisticated than an absorption (transmission) experiment, they provide a wealth of the information about the molecular structure, interaction and dynamics of species.