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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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cam967

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专业: 高分子，纳米，生物

活细胞单分子成像

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tong5028(金币+12):THANK YOU~~ 1-17 23:04

因为QDs(量子点超敏荧光试剂盒)光褪色临界值高，激发和发射光谱容易分离，所以它的信噪比很高，非常适于长期追踪单分子。
但是，由于QDs光稳定性好，许多用于区分信号是否来自单个荧光团的方法（如一步光褪色法）是不能用于QD的。
另一种检测方法-荧光闪烁法，也因QD荧光闪烁对激发密度，温度，和周边环境都很敏感无法有效检测。这些因素有时甚至会使荧光闪烁消失，比如在还原性环境中。
而细胞环境具有很高还原性，所以荧光闪烁法是不合适的。
此外，和有机化合物不同，他们的闪烁间隔只有0.5ms，QD的闪烁间隔在500ms左右。事实上，已经商业化的抗生物素蛋白-QD的闪烁间隔为100s.这么长的闪烁间隔会使QD无法跟踪细胞质中的单分子，因为单分子的扩散速度为3 mm2/s
上述这些因素，使确定单分子开始发射信号的时间成为难题。
目前生物-QD由于具有多重化合价，使的QD在活性细胞标记单分子变得更加困难。这是目前研究单分子的最大障碍，因为多化合价会影响分子的行为甚至功能。
不过，由于QD可以避免光破坏和金属降解，量子产额高，以及跟踪单分子却不影响单分子功能的潜力，激励我们去开发QD，并采用新的方法去克服这些弱点