Biomedical Engineering Reference
In-Depth Information
This increased sensitivity, combined with unprecedented photostability, has allowed
for single proteins to be dynamically tracked in live cells studies (Dahan et al.
2003
;
Lidke et al.
2004
) .
QDs possess a broad excitation that favors excitation at low (blue) visible wave-
lengths. Unlike conventional organic dyes, the QD emission spectrum is symmetri-
cal and quite narrow (30-40 nm) and can be precisely tuned by varying QD size
(Bruchez et al.
1998
; Michalet et al.
2001
). Furthermore, the Stoke's shift, the sepa-
ration between excitation and emission wavelengths, of QDs is large compared to
that of organic dyes and further enhances the ability for multicolor imaging.
Altogether, this allows QDs to serve as fl uorescent tags for a wide spectrum of col-
ors, which can be excited with a single wavelength or excitation source and can be
viewed simultaneously. Indeed, QD barcode technology has demonstrated that by
multiplexing 5-6 QD colors at six intensity levels in polymer beads, it would be
possible to encode among thousands of multiple emissions colors (Han et al.
2001
) .
While any one of the above QD photophysical properties would alone be a marked
improvement over conventional organic dyes, taken together they represent a dra-
matic leap forward in fl uorescent imaging technology.
There are some new photophysical properties of QDs that are not well under-
stood and have not been well examined in cellular applications. For example, QDs
undergo fl uorescence intermittency, or blinking, caused by surface defects in the
nanocrystal, which act as traps for electron-hole pairs and prevent their recombina-
tion (Nirmal and Brus
1999
; Yao et al.
2005
). Blinking “on” durations vary inversely
with excitation intensity while the “off” state is excitation independent and is depen-
dent on the QD coating and is present for QDs immobilized onto surfaces as well as
in aqueous physiological environments (Nirmal and Brus
1999
; Yao et al.
2005
) .
Remarkably, the blinking lasts on a several second time scale (0.1 s to ~10 min)
(Yao et al.
2005
). On a practical basis, blinking is a convenient signature of single
QDs. Since aggregates of QDs will cancel out this behavior through statistical coin-
cidence, the presence of intermittent fl uorescence in QD samples can serve as a
good indicator of single molecule labeling. However, because QD blinking can vary
in rate and is dependent on surrounding environment, if not well controlled, can
prove to be a problematic artifact of QD fl uorescence and interfere with applications
such as estimating dynamic single-molecular tracking at rapid timescales as well as
fl uorescence resonant energy transfer studies. In addition, blinking is correlated
with spectral jumping, or change in emission peak position (Pinaud et al.
2006
) .
Currently, reduction in blinking is being addressed by changing the composition of
the core and thickening of the outer shell, which it is believed will eliminate blink-
ing (Michalet et al.
2005
). The parameters that infl uence blinking in physiological
environments are just only beginning to be investigated. In the future, if blinking
can be well-controlled, this property may actually prove useful to sense localized
environmental conditions.
Another less investigated photophysical property of QDs is the infl uence of local
physiological environments on QD fl uorescence. QD fl uorescence is highly dependent
on environmental conditions. Spin-coated QDs exhibit changes in fl uorescence fl uctua-
tions when exposed to dry versus humid nitrogen environments (Michalet et al.
2001
)