Chemistry Reference
In-Depth Information
can be spent trying to overlap them perfectly. Alternatively, the lasers can be focused
at different spots, and
flow used to put molecules through both laser foci [37]. If
ne
measurements of centroids of
fluorescence are to be used to attain precise position-
ing of the molecules, this methodology will not work well (because of the chromatic
aberrationsmentioned above). When extending this concept tomore than two colors,
all lasers will need to be focused to the same focal volume.
One excitation, many colors
If possible, it is simpler to excite all colors simulta-
neously with a single laser. This could be accomplished in at least two ways. First,
FRET between a donor and acceptor dye may be used to detect the presence of the
acceptor-labeled component as long as it is bound to the donor-labeled component.
Unfortunately, in order for this method to work, donor and acceptor must be
relatively close to each other (within
2
-
8 nm).
Second, alternative dyes may be used. Examples include quantum dots [10] and
hybrid
uorophores that have extra large Stokes shifts [38]. Proof-of-principle of these
methods has been shown to work, and they will be of great interest in monitoring
interactions.
Multiple excitations
alternating laser excitations
Since it is possible to replace
multiple excitation lasers with a single excitation laser, it would seem that use of
multiple laser excitations would fade with time. However, the recent development of
alternating laser excitation (ALEX) promises to stop this from happening [26]. ALEX
allows the combination of FRET information with the co-localization information
available from molecules being in the same optically isolated volume.
In previous multiple laser excitation applications, both lasers were on continu-
ously. Emission from each color is separated and monitored individually. In the
example of RNAP and DNA, if the
fluorophores are close together and FREToccurs
from one
fluorophore on the protein (donor) to the
uorophore on the DNA
(acceptor) at high ef
ciency, it is not possible to distinguish between acceptor
emission from FRET and acceptor emission from direct excitation by the second
(red) laser. Additionally, the donor emission is quenched, and the observer may
incorrectly conclude that the RNAP and DNA are not bound, since only acceptor
emission can be observed.
Conversely, if the RNAP and DNA are bound, but outside the range of FRET, a
single excitation format would result in only donor emission. The observer would
incorrectly conclude that the RNAP and DNA are unbound, because only donor
emission can be observed.
ALEX allows the correct conclusion to be drawn in both cases by using time-
division and wavelength division multiplexing. Only one laser is on at a given time,
but the donor excitation and acceptor excitation lasers are alternated more rapidly
than the systemdiffuses through the optically isolated volume or the dynamics of the
system. When the donor excitation laser is on, any FRET that occurs can be easily
detected. When the acceptor excitation laser is on, the presence of the acceptor
fluorophore is interrogated, even if there is no FRET.
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