Biomedical Engineering Reference
In-Depth Information
quantum dots and used them to promote neuronal-like differentiation in cultured
PC12 cells (Vu et al.
2005
). Ultimately, these approaches could be used to visual-
ize and track functional responses in neurons. However, as with any new technol-
ogy, there are caveats. For example, Vu et al. reported that bNGF conjugated to
quantum dots had reduced activity compared to free bNGF. Other groups are
pushing the technology forward and providing new quantum dot-based tools.
Brinker and colleagues developed a technique to produce biocompatible water-
soluble quantum dot micelles that retain the optical properties of individual quan-
tum dots. These micelles showed uptake and intracellular dispersion in cultured
hippocampal neurons (Fan et al.
2005
). Ting and colleagues are developing a
modifi ed quantum dot-labeling approach that addresses the relatively large size of
antibody-quantum dot conjugates and the instability of some quantum dot-ligand
interactions. Their technique tags cell surface proteins with a specifi c peptide (a
15 amino acid polypeptide called acceptor protein) that can be directly biotiny-
lated as a target for streptavidin-conjugated quantum dots (Howarth et al.
2005
) .
Using this approach, they were able to specifi cally label and track a-amino-3-hy-
droxyl-5-methyl-4-isoxazole-propionate (AMPA) receptors on cultured hip-
pocampal neurons.
3
Specifi c Labeling and Imaging of Dissociated
Neurons and Glial Cells
We have previously discussed in detail our quantum dot-labeling protocols for label-
ing neurons and glia (Pathak et al.
2006
). We conjugated anti-b-tubulin III and anti-
glial fi brillary acidic protein (GFAP) antibodies to 605-nm quantum dots and labeled
primary cortical neurons, PC12 cells, primary cortical astrocytes, and r-MC1 retinal
Muller glial cells. b-tubulin III and GFAP are ubiquitous cytoskeletal proteins spe-
cifi c to neurons and macroglia, respectively, but the protocols should label any pro-
tein of interest. Table
1
summarizes the detailed methods.
Using our protocols, we were able to get excellent specifi c labeling of b-tubulin
in neurons and PC12 cells and GFAP in astrocytes and Muller cells, with negli-
gible nonspecifi c binding or background (see Fig.
2
). Labeling with unconjugated
or primary antibody-omitted, streptavidin-conjugated quantum dots showed no
labeling at all (data not shown). b-tubulin and GFAP labeling using functionalized
quantum dots displayed similar labeling patterns to those expected using standard
ICC controls visualized with fl uorophore-tagged secondary antibodies (Fig.
2g, h
).
For comparable imaging conditions, quantum dot-labeled cells were brighter and
displayed more detailed and sharper microstructural anatomy. The pattern of
quantum dot labeling was typical for that observed in other cell types, displaying
a dense punctuate pattern and fi ne details of both intracellular intermediate fi la-
ments and cellular processes, unlike traditional fl uorophores which tend to have a
diffused appearance due to the broad point spread function of their fl uorescence
signal. Nonspecifi c artifact labeling using some quantum dot protocols may label
