When photons are absorbed by a fluorescent compound (Fig. 1A), within femtoseconds it undergoes a transition from a singlet ground state (So) to an excited state which can be S1 (thick line) or a higher vibrational energy level of S1 (denoted by the thin lines above S1). The excitation maximum of a particular fluorescent compound is the wavelength at which this process occurs most efficiently. Within picoseconds, molecules in higher vibrational states relax to the S1 state (thick line).

Most fluorescent molecules remain in the S1 state for 1-10 nsec on average, after which they can return to the S0 ground state through one of a few different relaxation pathways.[1] The radiative relaxation pathway occurs through emission of fluorescent light by the excited fluorophore as it returns to the ground state, indicated by the wavy arrow in Fig. 1A. The light emitted is always of lower energy (longer wavelength) than that of the exciting light.

Several nonradiative pathways compete with fluorescence emission for return to the ground state. These nonfluorescent pathways (labeled NF in Fig. 1A) include internal conversion (a return to S0 accompanied by release of heat), intersystem crossing (change in the electron-spin orientation resulting in relaxation to a long-lived triplet excited state before decaying to S0), and collisional deactivation (involving direct encounters between the dye and neighboring solvent molecules). These deactivation pathways are inherent to fluorescent dyes in solution and are present to various extents.

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