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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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