Chemistry Reference
In-Depth Information
may be determined within the limitations imposed by resolution of the detection
method, mobility of the emitter and binning (integration) time. This localization
accuracy can be very high (
1 nm) [3], allowing accurate tracking of the
uorophore-
and what it is attached to. Also, point emission from the
fluorophores allows
single-molecule spectroscopy since isolation of molecules is possible. Precise
positioning of the
fluorophore requires isolation.
Another important consequence is that, if two spectrally separable
uorophores
(different colors) are used, the relative position of those molecules attached to two
fluorophoresmay be obtained. The relative position and association can bemeasured
using co-localization, for molecules immobilized on surfaces or in a matrix, and
cross-correlation measurements, for freely diffusing molecules. The different colors
allow each
fluorophore to be isolated even when in close proximity [10]. More
recently, it has been shown that even if the
fluorophores are of the same color, but can
be turned on or off at different times, the same high accuracy resolution and even
imaging can be achieved [11].
9.2.3
Fluorescence Polarization-Measures Rotational Movement and Freedom of Movement
Fluorophores do not absorb or emit radiation uniformly in all directions. They act as
electric dipoles, which preferentially absorb and emit radiation that is aligned with
this dipole. The orientation of the
fluorophore plays a role in both absorption and
emission processes, due to their dipolar nature. The excitation rate k
e
(Figure 9.1A)
depends on the incident power, absorption cross-section
and relative orientation of
the incident electromagnetic
eld
E
and the absorption dipole moment
s
m
abs
:
!
!
abs
2
Þ
As mentioned, the emitted intensity is not only proportional to the population of the
excited state S
1
, but also to the detection ef
ciency which can be chosen to be
polarization sensitive. Polarization-sensitive (time-resolved) measurements can thus
yield information on the (time-dependent) orientation of the
uorophore, and have
been used to study DNA and protein conformations at the single-molecule level as
will be reviewed later [12
k
e
¼ sj m
j
ð
9
:
3
15].
-
9.2.4
Fluorescence Resonance Energy Transfer-nm-scale Ruler
Fluorescence resonance energy transfer (FRET) is one of the primary tools used in
single molecule spectroscopy, allowing measurements of distances between 2 and
8 nm at the single-molecule level [16
-
19]. Some prefer to call it Forster resonance
energy transfer, after the scientist who
first described it, primarily because FRET
involves the non-radiative transfer of energy from one
uorophore to another. They
feel that since the process is non-radiative, the appellation
uorescence is inap-
propriate. However, in order for the non-radiative transfer to occur, the
uorescence
emission spectrum of a donor (D)
fluorophore must overlap the
uorescence
