Biomolecular and Bioanalytical Techniques

14 Fluorescence Spectroscopy and Its Applications in Analysing Biomolecular Processes

Nathan N. Alder

Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT, 06269, USA

14.1 Significance and Background

Luminescence is the process by which molecules emit light from electronically excited states that result from chemical reactions, mechanical forces or the absorption of electromagnetic ( EM ) radiation. Photoluminescence occurs when the excited state originates from the absorption of photons in the ultraviolet (UV) or visible region of the EM spectrum, a process called photoexcitation. The process of photoluminescence encompasses two phenomena, fluorescence and phosphorescence, which differ based on the electronic configuration of the excited state.

Fluorescence can be described as a series of photophysical events. First, a fluorescent molecule (fluorophore) in its ground state absorbs a photon and shifts to a high‐energy excited electronic state on a picosecond (10⁻¹⁵ seconds) timescale. The fluorophore then occupies this excited state on the order of nanoseconds (10⁻⁹ seconds) before relaxing back to the ground state with the concurrent emission of a fluorescent photon. We can relativise this process to a more relatable timeframe: if the time for photon absorption and excitation were extended to one second, then the time required for the emission of fluorescent light would be several days. This comparison illustrates that during the fluorescence ...