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In-Depth Information
Excited singlet state
5
S
2
2
1
0
Internal co
nversion
3
5
S
1
Intersystem
Intersystem
crossing
2
Excited
triplet
state
1
crossing
3
5
0
T
1
2
1
0
Absorption
Absorption
(excitation)
Fluorescence
Fluorescence
(emission)
(excitation)
(emission)
Phosphorescence
Phosphorescence
(emission)
3
5
(emission)
S
0
2
1
0
Ground state
Figure 4.1 Jablonski energy diagram and Stokes shift.
singlet states (S
1
,S
2
), and the excited triplet state (T
1
), and the resulting
fluorescence and phosphorescence emission (
Fig. 4.1
).
Most aromatic molecules with delocalized electrons are theoretically able
to undergo luminescence and fluorescence phenomena. They can be associ-
ated to different sources in biological molecules, from natural intrinsic fluo-
rescent probes (tryptophan or natural fluorescent protein; GFP, RFP, etc.) to
small synthetic chemical dyes (Cyanine, Alexa, Atto, etc.). Fluorescence
technology constitutes an ideal noninvasive approach tomonitor and charac-
terize in detail specific interactions between biological molecules. A large
number of interactions can be investigated at both steady-state and kinetic
levels using either intrinsic or extrinsic fluorescence probes. Depending on
the type of interaction and the context, several fluorescence-based methods
are available, including solvatochromism, anisotropy, and fluorescence reso-
nance energy transfer (FRET) (
Fig. 4.2
). This chapter focuses on the different
applications of fluorescence technology to monitor specific events in biology
for both fundamental and mechanistic issues.
1.2. Solvatochromism and resonance energy transfer
1.2.1 Solvatochromism
Most fluorescent molecules can be considered environmentally sensitive
probes since there are several environmental parameters that can affect their
fluorescent properties. These environmental factors include the solvent,
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