Chemistry Reference
In-Depth Information
designed that display enhanced photostability in comparison to first generation
fluorophores such as fluorescein. Simultaneously, due to technical improvements,
readout times for many fluorescence techniques could be decreased. Despite these
improvements, the nevertheless limited photostability of organic chromophores can
still hamper microscopic applications requiring high excitation light intensities in
the UV/vis region or long-term imaging. Thus, the search for brighter and espe-
cially more stable dyes is still going on. With respect to photochemical stability,
lanthanide chelates can be superior to conventional organic chromophores.
In contrast, almost all types of luminescent nanoparticles display excellent
thermal and photochemical stability. From the range of these nanocrystals, QDs
are the ones most sensitive to photooxidation and photobleaching, but even these
effects can be almost completely suppressed by epitaxical growth of a protective
shell to shield the core material for relevant time intervals [
80
]. Moreover, the
inorganic nature of the QDs makes them typically resistant to metabolic degrada-
tion in live cells and organism which is beneficial, e.g., for long-term imaging. This
is a significant advantage over organic fluorophores for imaging applications, where
excitation with intense lasers is employed for long periods of time [
64
]. A superior
long-term stability compared to organic dyes has been demonstrated for example
for CdSe/ZnS and rhodamine-labeled tubulin [
42
] CdSe and Texas Red [
81
] as well
as for antibodies labeled with CdSe, FITC, R-phycoerythrin, and AlexaFluor 488
[
77
]. However, nanoparticles can show specific phenomena such as photobrighten-
ing [
82
] see also Sect.
3.7
on
Reproducibility, Quality Assurance, and Limitations
,
and undesired aggregation of nanocrystals can contribute to reduced stability.
The thermal and photochemical stability of both organic dyes and nanocrystals
are influenced by an extremely broad variety of conditions that need to be consid-
ered: excitation wavelength and intensity, matrix or microenvironment, label con-
centration, and, in the case of nanoparticles, surface chemistry. Therefore, the
individual study of the stability of a chromophore under the conditions required
can usually not be avoided.
2.4 Cyto- and Nanotoxicity
“All things are poison and nothing is without poison, only the dose permits
something not to be poisonous (Paracelsus).” Although this property of molecular
and nanoparticular reporters is not relevant for ex vivo applications such as
immunoassays, it is critical for imaging in cells or in vivo. In general, toxicity of
organic dyes is not often reported as a significant problem, with the exception
of DNA intercalators. Despite the ever increasing interest in in vivo imaging
applications and the obvious importance of cytotoxicity data of fluorescent repor-
ters for in vivo applications, there are only very few data available on the cytotox-
icity of NIR fluorophores at present [
78
,
83
].
The only organic fluorophores approved by the Food and Drug Administration
(FDA) for use in humans are fluorescein (e.g., for opthalometry), Nile Blue, and
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