Chemistry Reference
In-Depth Information
signal-relevant optical properties of fluorophores to be ideally insensitive to envi-
ronmental factors [
115
]. This renders the assessment of the sensitivity of chromo-
phores to their application-relevant environment increasingly important.
In addition, the photochemical stability of fluorophores also responds to dye
microenvironment.
The chromophore environment can affect the spectral position of the absorption
and emission bands, the absorption and emission intensity (
F
f
), and the fluores-
cence lifetime as well as the emission anisotropy, e.g., in the case of rigid matrices
or hydrogen bonding. Changes in temperature typically result only in small spectral
shifts, yet in considerable changes in the fluorescence quantum yield and lifetime.
This sensitivity can be favorably exploited for the design of fluorescent sensors and
probes [
24
,
51
], though it can unfortunately also hamper quantification from simple
measurements of fluorescence intensity [
116
]. The latter can be, e.g., circumvented
by ratiometric measurements [
24
,
115
].
The microenvironment dependence of the optical properties of organic fluoro-
phores is controlled by dye class, nature of the emitting state(s), excited state redox
potential, charge, and hydrophilicity. Dyes with resonant emission such as fluor-
esceins, rhodamines, and cyanines typically show only moderate changes in their
spectral characteristics, yet can change considerably in fluorescence quantum yield
and lifetime. Moreover, they are prone to aggregation-induced fluorescence
quenching (due to, e.g., homo-FRET and static quenching [
24
,
117
]. CT dyes
with an emission from an excited state that has a considerable dipole moment
like coumarins respond with notable spectral changes to changes in microenviron-
ment polarity as well as with changes in absorption and emission intensity. These
dyes can also be sensitive to solvent proticity. CT dyes, that are occasionally termed
solvatochromic dyes, can be thus exploited for the design of fluorescence probes for
microenvironment polarity [
118
].
In the case of QDs, the chromophore microenvironment mainly affects the
fluorescence quantum yield and fluorescence decay behavior. These effects are
governed by a whole range of factors: the nature of the nanocrystals, their ligands,
shells, and the accessibility of the core surface [
119
]. Typically, properly shelled/
ligated nanocrystals are minimally sensitive to microenvironment polarity provided
that no ligand desorption occurs [
5
]. Also, the emission and absorption properties of
most nanoparticles are barely responsive to viscosity, contrary to that of many
organic dyes. All nanoparticles are colloids and thus susceptible to changes in ionic
strength: electrostatically stabilized particles tend to aggregate upon increasing
ionic strength. Some nanoparticles (e.g., gold nanoparticles) are prone to aggrega-
tion-induced optical changes that can be exploited as signal amplification strategy.
For both organic dyes and QDs, bioconjugation often leads to a decrease in
fluorescence quantum yield and thus typically also in emission lifetime. Parameters
that can affect label fluorescence are the chemical nature and the length of the
spacer and, at least for organic dyes, the type of neighboring biomolecules like
oligonucleotides or amino acids in the bioconjugated form.
Generally, the knowledge of microenvironment effects greatly simplifies label
choice. This is an advantage of organic dyes as the spectroscopic properties of many
e
M
,
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