Biophotonics_Concepts_to_Applications

(Dana P.) #1

As Sects.6.7and8.4describe, certain types offluorophores within molecules
have the ability to absorb external excitation light at a particular wavelength, thereby
boosting the molecule to a higher-energy excited state. In general, this excited state
is energetically unstable and the molecule rapidly relaxes from the excited state back
to the ground state. This relaxation can take place through processes such as energy
dissipation into heat, internal transitions between different excitation states of the
molecule, or by means of afluorescence process. During thefluorescence process,
thefluorophore will emit light of a longer wavelength (lower energy). The terms
singlet state (designated by S) and triplet state (designated by T) are used in
describing molecular energy states influorescence. In quantum mechanics, asinglet
statehas only one allowed value of the spin component, which is 0. Atriplet state
can have three allowed values of the spin component, which are−1, 0 and +1.
A genericfluorescence process is illustrated in Fig.9.2. First a molecule can
absorb incoming light, which occurs on the order of 10−^15 s (femtoseconds). As
indicated by the solid upward arrows, this action elevates the molecule from the S 0
ground state to a higher vibrational singlet energy level such as S 2 , according to
quantum mechanical transition rules. Immediately following the absorption, the
molecule drops to the lowest more stable vibrational energy level (theν= 0 level)
within the S 2 state by means of a nonradiative transition shown by the dashed
downward arrows. This transition occurs on the order of 10−^12 s and is called a
vibrational relaxation.
Subsequently, because theν= 0 level within the S 2 state is very close in energy
to an excited vibrational level of the S 1 state, a rapid energy transfer occurs between
the S 2 and S 1 states. The wavy horizontal line shows this process. This process is
referred to asinternal conversionand takes place on the order of 10−^13 s. Next
another vibrational relaxation process takes place from the excited vibrational level
to the lowest more stableν= 0 vibrational energy level of the S 1 state. This process
occurs on the order of 10−^12 s.


Emission wavelength (nm)

400 450 500 550 600

4

3

2

1

Fluorescence intensity (arbitrary units)

Normal tissue

Diseased tissue

Fig. 9.1 Generic examples
of the differences in
fluorescence spectra of
normal and diseased tissues


262 9 Spectroscopic Methodologies

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