6.7 Fluorescence Basics
Fluorescenceis the property of certain atoms and molecules to absorb light at a
particular wavelength and, subsequently, to emit light of a longer wavelength after a
short time [ 61 – 63 ]. Thefluorescence process is characterized by three key events,
all of which take place on time scales that are separated by several orders of
magnitude. Thefirst event generally involves using ultraviolet light (λ≤300 nm)
to excite molecules to higher vibrational states. This transition occurs in fem-
toseconds (10−^15 s). In the second event, the molecules then relax to slightly lower
excited energy states in the order of 10−^13 s. Subsequently, in the third event, the
state of the molecule transitions to a lower ground-state level in the order of 10−^9 s.
This third time interval is called thefluorescence lifetime. A photon of lower energy
(longer wavelength) than the excitation energy is emitted during the transition to the
ground state. Thisfluorescence process can be used to analyze the characteristics of
the molecule or to observe how the molecule interacts with other molecules.
Thefluorescence process is illustrated in Fig.6.29, which is called aJablonski
diagram. Here the label S 0 is the ground state energy with different vibrational
energy levels shown by closely spaced dashed horizontal lines. The labels S 1 and S 2
are thefirst and second excited states, again with different vibrational energy levels
shown by closely spaced dashed horizontal lines. Energy bands to which transitions
are forbidden separate the ground state and the excited states. Transitions between
the states occur only in terms of quantized energy units, that is, photons. The solid
vertical lines represent either absorption of an excitation photon (rising line) or
emission of afluorescent photon (dropping line). The dashed vertical lines represent
nonradiative internal relaxation process whereby some energy is converted to heat.
In the absorption process, an incoming photon (λ 1 orλ 2 ) will excite the molecule
to some upper vibrational level of thefirst or second excited state. Subsequently, the
molecule will rapidly relax through a nonradiative internal process to the lowest
level of S 1. Then from this S 1 level the molecule will relax to the S 0 ground state,
which results in afluorescent photon being emitted (λ 3 orλ 4 ).
Fluorescent substances are known asfluorophores. These can be broadly clas-
sified as intrinsicfluorophores and extrinsicfluorophores.Intrinsicfluorophoresare
biological molecules that canfluoresce naturally through their interaction with
incident excitation photons. This process is known asautofluorescenceornatural
fluorescence. Examples of intrinsicfluorophores are the aromatic amino acids (e.g.,
Table 6.5 Excitation and
fluorescence wavelengths of
some common intrinsic
fluorophores
Molecule Excitation (nm) Fluorescence (nm)
Tryptophan 280 300 – 350
Tyrosine 270 305
Flavin 380 – 490 520 – 560
Collagen 270 – 370 305 – 450
Melanin 340 – 400 360 – 560
NADH 340 4506.7 Fluorescence Basics 189