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Unformatted text preview: ® Molecular Probes Molecular Probes The Leader in Fluorescence Technology Introduction to Fluorescence Techniques ing, fluorescence energy transfer and intersystem crossing (see below) may also depopulate S 1 . The fluorescence quantum yield, which is the ratio of the number of fluorescence photons emitted (Stage 3) to the number of photons absorbed (Stage 1), is a measure of the efficiency of fluorescence in competition with these other processes. Stage 3 : Fluorescence Emission A photon of energy h ν EM is emitted, returning the fluorophore to its ground state S . Due to energy dissipation during the excited- state lifetime, the energy of this photon is lower, and therefore of longer wavelength, than the excitation photon h ν EX . The difference in energy or wavelength represented by (h ν EX –h ν EM ) is called the Stokes shift. The Stokes shift is fundamental to the sensitivity of fluorescence techniques because it allows emission photons to be detected against a low background, isolated from excitation pho- tons. In contrast, absorption spectrophotometry requires measure- ment of transmitted light relative to high incident light levels at the same wavelength. Fluorescence Spectra The entire fluorescence process is cyclical. Unless the fluoro- phore is irreversibly destroyed in the excited state (an important phenomenon known as photobleaching, see below), the same fluorophore can be repeatedly excited and detected. For polyatomic molecules in solution, the discrete electronic transitions represented by h ν EX and h ν EM in Figure 1 are replaced by rather broad energy spectra called the fluorescence excitation spectrum and fluorescence emission spectrum, respectively. The bandwidths of these spectra are parameters of particular importance for applications in which two or more different fluorophores are simultaneously detected (see below). With few exceptions, the fluorescence excitation spectrum of a single fluorophore species in dilute solution is identical to its absorption spectrum. Under the same conditions, the fluorescence emission spectrum is independent of the excitation wavelength, due Introduction to Fluorescence Techniques Figure 1. Jablonski diagram illustrating the processes involved in the creation of an excited electronic singlet state by optical absorption and subsequent emission of fluorescence. The labeled stages 1, 2, 3 are referred to in the text. Stage 1 : Excitation A photon of energy h ν EX is supplied by an external source such as an incandescent lamp or a laser and absorbed by the fluorophore, creating an excited electronic singlet state (S 1 ′ ). This process distin- guishes fluorescence from chemiluminescence, in which the excited state is created by a chemical reaction....
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- The Land, molecular probes, Fluorophores