Chemistry Reference
In-Depth Information
It does require expensive laser and other components. SAF requires a water
-
glass
interface and special objectives, but provides very small volumes.
Although thesemethods are critically important for the single-molecule analysis of
interactions, they still need further development to be widely applicable.
9.3.3.2 Multiple Points
TIR
In order to look at many molecules at one time, an alternative optical isolation
method is commonly used. Total internal re
ection is 100% re
ection obtained at a
dielectric interface when the approaching light beam is contained inside a medium
with a higher index of refraction than that of the medium on the other side of the
interface and the angle of incidence is larger than a critical angle. Light incident on a
glass
-
water interface can undergo total internal re
ection for
60
from the normal
to the interface. When a light beam is re
ected under these conditions, it is 100%
re
ected, but an evanescent (exponentially decaying) beam does penetrate the lower
index of the refraction medium, in this case the water. This evanescent wave can
excite
uorophores near the surface of the glass (within
>
150 nm), but does not excite
molecules which are deeper in the water. This provides optical isolation without a
pinhole. Total internal re
ection can be obtained over a wide area (50
m
2
or
more), allowing excitation and optical isolation of many single molecules simulta-
neously. This is a great help in saving experimental time. The main drawback of this
technique is that the molecules are required to be at the glass
-
water interface.
50
m
Multi-confocal spectroscopy Multiple spot techniques have been demonstrated or
proposed in a few papers that provide a glimpse of how the single point measure-
ments could be extended to multiple simultaneous points. Two-point cross-correla-
tion techniques have been used to monitor systems undergoing
ow [35]. The zero-
mode waveguides can be patterned in arrays that may be simultaneously moni-
tored [32]. Cubic lattices of foci have been proposed for rapid multi-confocal
analysis [36].
9.3.3.3 How many Excitation Lasers?
If the proposed studymonitors only
fluorophores of one color, this question is easy to
answer
a single laser should be used. However, most studies of protein dynamics,
and especially interactions, will require more than one color and one
uorophore to
address the scienti
c questions. For instance, consider a labeled RNA polymerase
attached to a labeled DNA fragment. In order to verify that RNAP is actually
interacting with the DNA, it is necessary to see both the RNAP and the DNA in
the same optically isolated volume. This requires two colors in order to distinguish
between the two components.
The most straightforward method of obtaining multiple colors is to excite two
fluorophores of different colors with two lasers, one for each color. If the optical
isolation provided for both wavelengths overlap, then co-localization of
uorescence
for both colors indicates binding of the twomolecules. The problem is that, especially
in the confocal case, the optical excitations do not overlap perfectly, and much time
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