Reynolds
14
maxima of the absorption and emission spectra of the same electronic transition, the socalled Stokes Shift. There are three fundamental processes that are implicit in the emission
of light by a molecule (Lakowicz, 2006): excitation of the molecule (i.e., absorption of an
appropriate photon), vibrational relaxation (nonradiative decay), and finally the emission
of light (radiative decay). All of these processes occur on timescales that are separated
by several orders of magnitude (see Table 1.2). Excitation of a molecule by an incoming
photon happens instantaneously (femtoseconds, 10E
–15
), while the vibrational relaxation
of an electron in an excited state to the lowest energy level is slower, usually occurring
over picoseconds (10
–12
). The final process, light emission, almost always occurs at longer
wavelengths and the return of the molecule back to the ground state occurs over nanoseconds (10
–9
seconds).
1.3.1 Electronic Transitions
The interaction between a molecule and optical radiation manifests itself in various radiative and nonradiative processes as shown in Figure 1.3. Many of the colors that we experience in everyday life (flowers, green vegetation, synthetic dyes, etc.) are the result of the
transition of an electron from one electron orbital into another. Sigma electrons require
high energies found only at very short wavelengths in the vacuum UV (100 nm–200 nm)
if they are to be promoted to an available molecular orbital at a higher energy level. They
are of little relevance to most fluorescence techniques, which are usually concerned with
wavelengths between 200 nm and 1000 nm. Pi electrons are less tightly bound to the nuclei
than σ electrons. As a result, the energies required for ionization and electronic transition are lower than for σ electrons but still mainly in the vacuum UV or the middle UV
(200 nm–300 nm). However, for delocalized π electrons in conjugated systems the energies required for electronic transitions are much lower and easily obtained in the near
UV (300 nm–400 nm). For extensively delocalized systems or super-delocalized systems
electronic transitions can occur at energies ranging from the near UV to the near infrared
(200 nm–1500 nm). The preference of a particular process over another is dictated by the
energy of the “exciting” field, that is, the wavelength of the light, the configuration of the
molecule(s), and its environment.
1.3.1.1 Spin Multiplicity
The electronic state of a molecule determines both the distribution of negative charge and
its overall geometry. All molecules exhibit different electronic states (illustrated as S 0 , S 1 ,
and S 2 in Figure 1.3), depending on the total electron energy and the symmetry of various electron spin states. Each electronic state contains a number of vibrational and rotational energy levels associated with the atomic nuclei and bonding orbitals. As discussed
in Section 1.2.1.2, electrons are described within a particular electronic state by their spin
quantum number (s), with values ms = +½ or –½. In Figure 1.4 the different spin states are
shown in terms of their orientation, spin-down or spin-up, with the direction of the arrows
14
maxima of the absorption and emission spectra of the same electronic transition, the socalled Stokes Shift. There are three fundamental processes that are implicit in the emission
of light by a molecule (Lakowicz, 2006): excitation of the molecule (i.e., absorption of an
appropriate photon), vibrational relaxation (nonradiative decay), and finally the emission
of light (radiative decay). All of these processes occur on timescales that are separated
by several orders of magnitude (see Table 1.2). Excitation of a molecule by an incoming
photon happens instantaneously (femtoseconds, 10E
–15
), while the vibrational relaxation
of an electron in an excited state to the lowest energy level is slower, usually occurring
over picoseconds (10
–12
). The final process, light emission, almost always occurs at longer
wavelengths and the return of the molecule back to the ground state occurs over nanoseconds (10
–9
seconds).
1.3.1 Electronic Transitions
The interaction between a molecule and optical radiation manifests itself in various radiative and nonradiative processes as shown in Figure 1.3. Many of the colors that we experience in everyday life (flowers, green vegetation, synthetic dyes, etc.) are the result of the
transition of an electron from one electron orbital into another. Sigma electrons require
high energies found only at very short wavelengths in the vacuum UV (100 nm–200 nm)
if they are to be promoted to an available molecular orbital at a higher energy level. They
are of little relevance to most fluorescence techniques, which are usually concerned with
wavelengths between 200 nm and 1000 nm. Pi electrons are less tightly bound to the nuclei
than σ electrons. As a result, the energies required for ionization and electronic transition are lower than for σ electrons but still mainly in the vacuum UV or the middle UV
(200 nm–300 nm). However, for delocalized π electrons in conjugated systems the energies required for electronic transitions are much lower and easily obtained in the near
UV (300 nm–400 nm). For extensively delocalized systems or super-delocalized systems
electronic transitions can occur at energies ranging from the near UV to the near infrared
(200 nm–1500 nm). The preference of a particular process over another is dictated by the
energy of the “exciting” field, that is, the wavelength of the light, the configuration of the
molecule(s), and its environment.
1.3.1.1 Spin Multiplicity
The electronic state of a molecule determines both the distribution of negative charge and
its overall geometry. All molecules exhibit different electronic states (illustrated as S 0 , S 1 ,
and S 2 in Figure 1.3), depending on the total electron energy and the symmetry of various electron spin states. Each electronic state contains a number of vibrational and rotational energy levels associated with the atomic nuclei and bonding orbitals. As discussed
in Section 1.2.1.2, electrons are described within a particular electronic state by their spin
quantum number (s), with values ms = +½ or –½. In Figure 1.4 the different spin states are
shown in terms of their orientation, spin-down or spin-up, with the direction of the arrows