Biophotonics_Concepts_to_Applications

Several relaxation transition possibilities then can take place to return the
molecule back to the S 0 state from the S 1 state. The two main ones are a radiative
transition wherein afluorescence photon is emitted or there can be anintersystem
crossingto an excited level in thefirst triplet state T 1. Thefluorescence emission
takes place from the lowest vibrational level of the singlet S 1 state, as indicated by
the solid downward arrows going to the S 0 state. Thisfluorescence occurs on the
order of 10−^9 s. The transition from the excited level to the lowest level in thefirst
triplet T 1 state also takes place by means of vibrational relaxation. The eventual
transition from the lowest level of the T 1 state to excited vibrational levels of the S 0
state are calledphosphorescenceand take place in times of 10−^3 – 102 s.

9.2 FRET/FLIM

The phenomenon of raising molecules to an excited state and then observing their
fluorescent characteristics are important methods for investigating the biological
and chemical properties of biological tissues and systems. This section describes
two widely used techniques in this discipline. These are Förster resonance energy
transfer andfluorescence lifetime imaging microscopy, which are popularly known
as FRET and FLIM, respectively.

9.2.1 Förster Resonance Energy Transfer

Förster resonance energy transfer(FRET) describes a process of energy transfer
between two light-sensitive molecules (fluorophores) [ 10 – 13 ]. This process also is
known asfluorescence resonance energy transfer(again designated by FRET),

S 0

Fluorescence

10 -3 -10^2 s

Energy levels for

excited state 2

Ground state

energy levels

10 -15 s 10 -9 s

Energy

E

S 2

ν= 0

ν= 2

ν= 1

ν= 3

ν= 1

ν= 3

ν= 2S 1

Energy levels for

excited state 1

ν= 1

νν= 3= 2

T 1

ν= 0

Absorption Phosphorescence

10 -12 s

10 -13 s

Vibrational relaxation

Internal conversion

Intersystem crossing

Fig. 9.2 Energy transition diagram of a genericfluorescence process

9.1 Fluorescence Spectroscopy 263