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visible light is the part of the electromagnetic spectrum that ranges from 400 to 700 nm. a common source of light for photochemical studies is the sun. for many years, most photochemical reactions were carried out using continuous light from a variety of sources. a more recent trend is the development of the microchip spectrophotometer, which allows detailed examination of photochemical reactions under more defined and stable light conditions ( note 17.1 ). because these devices can be used to both study and perform chemical reactions in the presence of light, they have become useful for photochemical studies in the pharmaceutical industry.
the fluorescence quantum yield (fqy) is a measure of the efficiency with which a fluorophore emits light when it is excited by a certain energy source. the fluorescence quantum yield is expressed as a proportion and is equivalent to the radiative efficiency of the fluorophore. the fluorescence quantum yield is usually expressed as a percentage.
synthetic dyes with emission wavelengths in the visible region are of particular interest for intracellular imaging because of the feasibility of light sources capable of exciting the desired wavelength. with the advent of the laser technology, fluorescent dyes with absorption and emission wavelengths in the visible region are now available in a wide range of spectral bandwidths. an appropriate combination of emission and absorption spectra should include dyes that are excited by near-uv (350–550 nm) and red (615–675 nm) light.
when two or more fluorophores are excited at the same time, fret (fluorescence resonance energy transfer) between the fluorophores can be observed. the transfer of energy from one fluorophore to another (acceptor) can be detected by fluorescence if the emission spectrum of the acceptor overlaps the emission spectrum of the donor. fret has been demonstrated for energy transfer from donor molecules to acceptors such as protein-bound and covalently linked dyes ( figure 14 ) or free dyes in close proximity ( figure 15 ). 2516d825b2