Biomedical Engineering Reference
In-Depth Information
used techniques such as radiolabeling or immunohistochemical techniques suffer
from the drawbacks of limited spatial-temporal resolution and lack of real time
monitoring. Fluorescence imaging techniques currently offer the most information
about receptor dynamics; however, organic dyes provide only momentary visualiza-
tion of receptor distribution and movement due to rapid photobleaching effects
(
t
< 10 s). Moreover, photostable fl uorescent probes such as green fl uorescent pro-
tein (GFP) are poor fl uorophores for single molecule imaging as it is diffi cult to
determine if the fl uorescent signal is coming from the cell surface, the cell interior
or a combination of both these sites (Niemeyer
2001
; Vu et al.
2005
) .
Early applications of QDs to study receptor dynamics suggest that they will be a
signifi cant improvement over current fl uorescent tags. Dahan et al. used anti-glycine
antibody-conjugated QDs and confocal microscopy to track individual glycine recep-
tors in cultured spinal cord neurons (Dahan et al.
2003
). The small size of the anti-
body-conjugated QDs enabled access to the receptors in the synaptic cleft and provided
a signifi cant improvement over latex beads (200-500 nm in diameter) and colloidal
gold (40 nm in diameter) previously used to study receptor dynamics. The relatively
extreme brightness and photostability of the QD fl uorescence enabled the use of these
probes for tracking receptors in live cells over an extended time course of 20 min. In
addition, the blinking of these dots confi rmed the detection of individual receptors and
this property of QDs was used to follow receptor movement using time-lapse imaging.
The fl uorescence tracking study helped distinguish glycine receptors into three mem-
brane sub-domains based on their distinct diffusion properties.
Receptor mapping has also been demonstrated by Howarth et al. (Howarth et al.
2005
). A special feature of their study is that they observed glycine-evoked changes
in QD-tagged AMPA receptor distribution in hippocampal neurons transfected with
GluR1 or GluR2 subunits. GluR1 receptor sub-units were barely detectable before
the addition of glycine, but a signifi cant population was observed after application
of the glycine pulse. On the other hand, the GluR2 receptors showed little change in
distribution in response to glycine. Interestingly, they observed that the size of QDs
could limit their accessibility to a portion of GluR2 subunits. QD-tagged receptors
were detected co-localized with a chimeric post-synaptic density protein PSD95-
yellow fl uorescent protein (YFP). However, in experiments where the AMPA recep-
tor subunits were labeled with Alexa Fluor™ dyes instead of QDs, greater
co-localization with PSD95-YFP was found, than in the case of QD labeling.
In the future, new experiments using QD-tagged receptors will continue to con-
tribute to new knowledge concerning receptor spatial-temporal dynamics. In addi-
tion, receptor mapping using multiple QD colors will be useful for understanding
the interactions of multiple types of receptors and other synaptic proteins in live cell
preparations.
8.2
Quantum Dots as Ligand-Receptor Probes
Presynaptic neurons release ligands that act on the receptors of postsynaptic neu-
rons at close proximity as well as over longer distances. Where do these ligands go,
which subtype of receptors do they bind to, and how effective are they in binding to
