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ensembles of QDs, accurate quantification thus requires the ratio of emissive to
nonemissive QDs to be constant.
Generally, reliable and comparable fluorescence measurements require fluo-
rescent labels with reproducible physico-chemical properties and established
tools to evaluate this. This is a unique advantage of organic dyes. These com-
pounds can be synthesized on a large scale and characterized according to their
structure and purity using well-established analytical techniques. This is more
challenging for dye-biomolecule conjugates, such as fluorophore-labeled antibo-
dies or proteins, due to batch-to-batch variations in label density and label density
distribution and the lack of methods to reliably and accurately determine label
density. Nevertheless, this is manageable in principle. In the case of QDs, the
colloidal nature of these chromophores, in conjunction with the broad variety of
synthetic strategies and surface functionalities, renders chromophore characteri-
zation more challenging compared to organic dyes. For commercial QDs, this is
often further complicated by the fact that commercial distributors usually refrain
from providing any information about the ligand(s). For instance, at present, there
are no established methods available to determine the surface coverage and
number of ligands attached to the surface of a QD. Even more challenging is
the characterization of QD-biomolecule conjugates, e.g., the measurement of the
QD-to-biomolecule ratio [
143
].
4 Applications of Nanoparticles: State-of-the-Art
and Future Trends
Organic molecules are well established as fluorescent labels and reporters for
in vitro assays and in vivo imaging, despite their nonoptimum spectroscopic
features and photochemical instability. Due to their availability from many com-
mercial sources, established functionalization protocols, and extensively studied
properties organic dyes present a simple, safe, and comparatively inexpensive
option. This holds similarly true for metal ligand complexes and lanthanide che-
lates. To further improve the reliability of the data obtained with these labels and
reporters, e.g., the fluorescence quantum yields of typical chromophores under
commonly used measurement conditions should be reevaluated and comparative
photostability studies could be beneficial. With respect to the ever increasing
number of in vivo applications of chromophores, reliable data on the cytotoxicity
of these chromophores are also needed, preferably obtained under standardized
measurement conditions. Generally, there is an increasing need for bright and stable
NIR chromophores [
144
]. Whether this can be met with the rational design of
organic dyes, metal ligand complexes, and lanthanide chelates or whether the use of
established NIR chromophores encapsulated into organic or inorganic nanoparti-
cles is a more straightforward approach to tune the spectroscopic properties and the
stability of such NIR fluorophores [
145
] remains to be seen in the coming years.
Here, particulate labels and reporters are expected to have a bright future if the
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