Dye-Doped Polymeric Particles for Sensing and Imaging - Advanced Fluorescence Reporters in Chemistry and Biology - page 206

with simple and inexpensive instrumentation. Therefore, interrogation via decay
time is usually applied for oxygen-sensitive and temperature-sensitive beads which
rely on luminescent metal complexes. Autofluorescence from the sample can be
easily discarded in time resolved measurements.
Dual lifetime referencing (DLR) is another powerful technique that enables
referenced measurements in case of fluorescent indicators [ 23 ]. In this method,
the analyte-dependent signal from an indicator is referenced against the signal from
an inert luminophore. This can be realized in both the time domain [ 24 ] and in the
frequency domain [ 25 ]. Often, a luminescent reference dye is embedded into gas
blocking nanobeads to avoid oxygen quenching. Polymers with very low gas
permeability such as poly(acrylonitrile) [ 24 ] or poly(vinylidene chloride- co -acry-
lonitrile) [ 26 ] are the best choice here.
Microscopic imaging of various parameters (oxygen, pH, etc.) with the help of
dye-doped nanobeads has become more and more popular and is mostly realized
with help of conventional fluorescent microscopes. Confocal and two-photon
microscopies enable higher resolution and, therefore, are very popular. Since the
light intensities are rather high, only highly photostable indicators can be used for
microscopic applications in order to avoid drifts caused by photobleaching. Unfor-
tunately, most conventional indicators are poorly suitable for the application in
two-photon microscopy due to very low two-photon absorption cross-sections.
Therefore, design of more efficient luminescent dyes is of extreme importance
[ 27 ] for such applications and their application in nanobeads for sensing and
imaging is yet to be realized.
5 Selected Examples of Dye-Doped Polymeric Bead Materials
Dye-doped polymeric beads are commonly employed in different formats
(Fig. 5 ), namely as water-dispersible nanosensors, labels and in composite
materials (DLR-referenced and multianalyte sensors, sensor arrays, magnetic
materials, etc.). The sensing properties of the dye-doped beads are of little or no
relevance in some more specific materials, e.g., the beads intended for photody-
namic therapy (PDT). The different formats and applications of the beads will be
discussed in more detail in the following section, and the relative examples of
sensing materials will be given.
5.1 Water-Dispersible Nanosensors
These are introduced directly into the analyzed medium or even into the cells to
measure analytical parameters there. Compared to dissolved indicators nanosen-
sors are usually significantly less toxic and are less prone to interferences and
quenching. On the other hand, similarly to the dissolved indicators, nanosensors