105
lifetime) must be attributed to either changes in the pigment environment or to additional
processes competing to relax the excited state. In photosynthetic systems, the major process
competing for excited state energy is photochemistry. The photochemical reactions of
photosynthesis convert chI excited state energy into chemical energy in the form of oxidants
and reductants that ultimately drive electron transport and CO2 fixation.
In quantifying fluorescence emission, it is important to distinguish between fluorescence
(quantum) yield, 4>r. and fluorescence intensity (F). The fluorescence yield is a constant which
relates the rate of light absorption (lJ to the rate (intensity) of fluorescence emission:
(5)
Thus, for a given state of the sample, the fluorescence intensity F increases linearly with
incident light intensity while the fluorescence yield is independent of light intensity. Clearly,
fluorescence intensity and quantum yield are not equivalent parameters, although the terms
are often (incorrectly) used interchangeably.
The fluorescence emission spectrum of any pigment is quantitatively related to the absorption
spectrum for formation of the excited state from which the fluorescence originates. Thus for
chIs and related pigments, fluorescence arises only from the lowest energy red (620-680 nm)
absorption. There is no fluorescence emitted directly from the higher energy absorptions. The
absorption bands of photosynthetic pigments are broad, indicating that each electronic state
(ground and excited states) has associated with it a distribution of energies. This distribution
arises from thermally excited molecular vibrations in the pigment. At physiological
temperatures, a large number of these vibrational levels are accessible creating a broad
absorption. For both upward (absorption) and downward (fluorescence) transitions between
electronic states, the most probable starting point is near the lowest energy vibrational state
(Figure 3). However, the most probable end point for either transition is near the middle of
the distribution of vibrational states. The energy difference between the absorption and
fluorescence transitions is accounted for by thermal emission through the closely spaced
vibrational states. This loss of energy accounts for the wavelength difference between the
absorption and fluorescence emission maxima (the Stokes shift), which is in the range of 6-8
nm for chI.
lifetime) must be attributed to either changes in the pigment environment or to additional
processes competing to relax the excited state. In photosynthetic systems, the major process
competing for excited state energy is photochemistry. The photochemical reactions of
photosynthesis convert chI excited state energy into chemical energy in the form of oxidants
and reductants that ultimately drive electron transport and CO2 fixation.
In quantifying fluorescence emission, it is important to distinguish between fluorescence
(quantum) yield, 4>r. and fluorescence intensity (F). The fluorescence yield is a constant which
relates the rate of light absorption (lJ to the rate (intensity) of fluorescence emission:
(5)
Thus, for a given state of the sample, the fluorescence intensity F increases linearly with
incident light intensity while the fluorescence yield is independent of light intensity. Clearly,
fluorescence intensity and quantum yield are not equivalent parameters, although the terms
are often (incorrectly) used interchangeably.
The fluorescence emission spectrum of any pigment is quantitatively related to the absorption
spectrum for formation of the excited state from which the fluorescence originates. Thus for
chIs and related pigments, fluorescence arises only from the lowest energy red (620-680 nm)
absorption. There is no fluorescence emitted directly from the higher energy absorptions. The
absorption bands of photosynthetic pigments are broad, indicating that each electronic state
(ground and excited states) has associated with it a distribution of energies. This distribution
arises from thermally excited molecular vibrations in the pigment. At physiological
temperatures, a large number of these vibrational levels are accessible creating a broad
absorption. For both upward (absorption) and downward (fluorescence) transitions between
electronic states, the most probable starting point is near the lowest energy vibrational state
(Figure 3). However, the most probable end point for either transition is near the middle of
the distribution of vibrational states. The energy difference between the absorption and
fluorescence transitions is accounted for by thermal emission through the closely spaced
vibrational states. This loss of energy accounts for the wavelength difference between the
absorption and fluorescence emission maxima (the Stokes shift), which is in the range of 6-8
nm for chI.