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tong5028

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Single-molecule imaging in live cells
Owing to their high photobleaching threshold and the
large separation between the excitation and emission
spectra, QDs provide a very high signal-to-noise ratio,
making them suitable for the long-term tracking of single
molecules [12]. However, many of the approaches used to
distinguish whether or not a signal comes from a single
ﬂuorophore, such as single-step photobleaching, cannot be
applied to QDs owing to high photostability.
The use of another diagnostic approach, FLUORESCENCE
BLINKING of a single QD under continuous wave illumina-
tion, is also hindered by the sensitivity of QD blinking to
changes in excitation intensity [40], temperature [41] and
the surrounding environment [42]. These features alter
blinking characteristics to the extent of eliminating
blinking under some conditions, such as those found in a
reducing environment [43]. Because the cellular environ-
ment is highly reducing, it could potentially make
blinking a poor criterion for identifying single QDs in
live cells. In addition, unlike organic dyes, for which the
blinking interval is about 0.5 ms [44], for QDs this interval
is around 500 ms [40]. In fact, it has been recently reported
that this interval could be asmuch as 100 s for commercially
available avidin-conjugated QDs [43]. Such a long inter-
mittency will prevent the use of QDs to track single
molecules in the cytoplasm of live cells where they diffuse
at about 3 mm2
/s [45]. The above features limit the ability to
establish when a single QD molecule is being observed.
The multivalency of currently available QD bioconju-
gates further precludes their use for labeling only a single
molecule in live cells (Figure 2d,e). This is a major
impediment to studying single molecules because it can
result in a change in the behavior and even functionality
of the molecules. Nevertheless, the ability of QDs to avoid
photodamage and metabolic degradation, their high
quantum yield and the potential to develop approaches
to label single molecules without affecting their function-
ality provide the motivation to design QDs and to develop
imaging strategies that will overcome these limitations.

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