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\DOI{10.5802/crchim.294}
\datereceived{2024-01-04}
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\dateposted{2024-04-19}
\begin{document}

\begin{noXML}

%\makeatletter
%\def\TITREspecial{\relax}
%\def\cdr@specialtitle@english{French Women in Chemistry in 2024}
%%\def\cdr@specialtitle@french{Les chimistes fran\c{c}aises en 2024}
%\makeatother

\title{Dye-based fluorescent organic nanoparticles made from polar and
polarizable chromophores for bioimaging purposes: a bottom-up
approach}

\alttitle{Ing\'{e}nierie mol\'{e}culaire de nanoparticules organiques
fluorescentes pour la bioimagerie : d'une vision mol\'{e}culaire \`{a}
une vision collo\"{i}dale}

\author{\firstname{Jonathan} \lastname{Daniel}\CDRorcid{0000-0003-1317-3165}}
\address{Univ. Bordeaux, CNRS, Bordeaux INP, ISM, UMR 5255, F-33400 Talence, France}

\author{\firstname{Oph\'{e}lie} \lastname{Dal Pra}\CDRorcid{0009-0009-5388-7198}}
\addressSameAs{1}{Univ. Bordeaux, CNRS, Bordeaux INP, ISM, UMR 5255, F-33400 Talence, France}

\author{\firstname{Eleonore} \lastname{Kurek}\CDRorcid{0009-0002-7293-5334}}
\addressSameAs{1}{Univ. Bordeaux, CNRS, Bordeaux INP, ISM, UMR 5255, F-33400 Talence, France}

\author{\firstname{Chlo\'{e}} \lastname{Grazon}\CDRorcid{0000-0002-4564-8738}}
\addressSameAs{1}{Univ. Bordeaux, CNRS, Bordeaux INP, ISM, UMR 5255, F-33400 Talence, France}

\author{\firstname{Mireille} \lastname{Blanchard-Desce}\CDRorcid{0000-0002-1572-9545}\IsCorresp}
\addressSameAs{1}{Univ. Bordeaux, CNRS, Bordeaux INP, ISM, UMR 5255, F-33400 Talence, France}
\email[M. Blanchard-Desce]{mireille.blanchard-desce@u-bordeaux.fr}

\keywords{\kwd{Organic dyes}
\kwd{Luminescent nanoparticles}
\kwd{Two-photon absorption}
\kwd{Confinement effects}
\kwd{{In vivo} imaging}
\kwd{Stealthiness}
\kwd{Single particle tracking}}

\altkeywords{\kwd{Colorants}
\kwd{Nanoparticules luminescentes}
\kwd{Absorption \`{a} deux photons}
\kwd{Effets de confinement}
\kwd{Bioimagerie} 
\kwd{Furtivit\'{e}}
\kwd{Suivi de particules uniques}}

\thanks{Conseil general d'Aquitaine, University of Bordeaux, CNRS, 
Bordeaux-INP, ERC COMET (101077364)}

\dedicatory{Dedicated to the memory of Michel Vaultier}

\shortrunauthors

\begin{abstract}
In the last decades, inorganic nanoparticles have attracted growing
attention in the field of nanophotonics, especially for bioimaging
purposes. Among them luminescent metal-, semiconductor- or oxide-based
``hard'' nanoparticles have been the most widely used. Yet, they raise
concern with respect to toxicity and degradability issues. In that
context, we have developed innovative bottom-up approaches
towards ultrabright dye-based fluorescent organic nanoparticles
(dFONs). Our strategy is based on the design and synthesis of
custom-designed (multi)polar and polarizable dyes (PPDs) as building
blocks of dFONs. These nanoparticles are readily prepared using
expeditious and green protocols involving nanoprecipitation of the
hydrophobic dyes in water. Their luminescence can be tuned in the whole
visible region down to the Near Infra-Red I (NIR-I) region while their
nonlinear optical responses can be enhanced thanks to cooperative
effects. Intriguingly, the implemented strategy also enables modulating
and improving the dFONs colloidal and structural stability as well as
their photostability. As a result, dFONs made from PPDs that combine
unprecedented brightness (up to 
\tralicstex{10\tsup{8}\,M\tsup{-1}·cm\tsup{-1} and 10\tsup{6}\,GM}{$10^{8}\,\mathrm{M}^{-1}\cdot\mathrm{cm}^{-1}$ and $10^{6}$\,GM}),
remarkable colloidal stability and absence of toxicity, have been
elaborated, providing superior substitutes to Quantum Dots. Such dFONs
can be used as ultrasensitive contrast agents for {in vivo} two-photon
angiography in small animals, while hyper-bright NIR-emitting FONs,
that show remarkable photostability and excellent biocompatibility, can
be successfully imaged and tracked at the single particle level in
water. Furthermore, ultrabright dFONs of different colors that
internalize into cells can be tracked within living cells allowing
real-time multicolor single particles tracking. In contrast, stealth
emitters are required for tracking cell-surface receptors or exploring
the extracellular space. In this direction, spontaneously stealth,
size-tunable, ultrabright and red to NIR emitting dFONs were developed.
Thanks to these unique properties, these dFONs could be imaged and
tracked up to \tralicstex{150 µm}{$150~\upmu\mathrm{m}$} deep in brain tissue.
\end{abstract}

\begin{altabstract}
Au cours des derni\`{e}res d\'{e}cennies, les nanoparticules
inorganiques luminescentes ont suscit\'{e} beaucoup d'int\'{e}r\^{e}t
dans le domaine de la nanophotonique, tout particuli\`{e}rement en vue
d'applications en imagerie biologique. Parmi elles, les plus
populaires restent celles bas\'{e}es sur l'utilisation de m\'{e}taux,
de semi-conducteurs ou d'oxydes. Certaines de ces nanoparticules
soul\`{e}vent toutefois des risques li\'{e}s \`{a} leurs potentielles
toxicit\'{e} et leur impact environnemental reste toujours
d'actualit\'{e}. Afin de proposer une alternative tout-organique
\`{a} ces nanoparticules inorganiques, notre \'{e}quipe d\'{e}veloppe
des nanoparticules organiques fluorescentes (appel\'{e}es dFONs) issues
de l'assemblage mol\'{e}culaire de colorants sp\'{e}cialement
con\c{c}us pour obtenir des brillances comparables aux nanoparticules
inorganiques les plus brillantes. Notre strat\'{e}gie, qui s'appuie
sur une approche \textit{bottom}--\textit{up}, est bas\'{e}e sur
l'ing\'{e}nierie mol\'{e}culaire de colorants hydrophobes
particuliers, i.e.\ de chromophores (multi-)polaires et
polarisables (d\'{e}sign\'{e}s par le sigle PPDs), comme
\'{e}l\'{e}ments constitutifs des dFONs. Les dFONs \`{a} base de PPDs
sont facilement obtenues par nano-pr\'{e}cipitation dans l'eau des
colorants. L'approche d\'{e}velopp\'{e}e permet de moduler leurs
propri\'{e}t\'{e}s optiques, notamment leur fluorescence dans toute la
gamme du visible jusqu'au proche infrarouge (NIR-I), et d'augmenter
leurs r\'{e}ponses optiques non-lin\'{e}aires en exploitant et
contr\^{o}lant les effets de confinement. De fa\c{c}on \'{e}tonnante,
il est \'{e}galement possible de moduler les stabilit\'{e}s
collo\"{i}dales et chimiques des dFONs, ainsi que leur furtivit\'{e} ou
inversement leur capacit\'{e} \`{a} s'internaliser dans les cellules,
en jouant sur la structure des PPDs les composant. Nous avons pu
obtenir des dFONs pr\'{e}sentant des brillances \`{a} un et deux
photons exceptionnelles 
(\tralicstex{10\tsup{8}\,M\tsup{-1}·cm\tsup{-1} and 10\tsup{6}\,GM}{$10^{8}\,\mathrm{M}^{-1}\cdot\mathrm{cm}^{-1}$ and $10^{6}$\,GM}
respectivement), atteignant, voire surpassant, celles des
\textit{Quantum Dots}. L'absence de toxicit\'{e} des dFONs
associ\'{e}e \`{a} leurs brillances \'{e}lev\'{e}es et leur bonne
photostabilit\'{e} nous a permis de d\'{e}montrer leur pertinence comme
agents de contraste \textit{in vivo} pour l'angiographie ou le suivi
de particules individuelles \textit{in cellulo} ou dans l'espace
extracellulaire en profondeur dans le cerveau. 
\end{altabstract}

\maketitle

\clearpage

\twocolumngrid

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\section{Introduction} 


Fluorescence imaging has become a groundbreaking tool to study and
image biological environments and processes, in domains ranging from
medical applications to fundamental research in biology. In that
context, a wide variety of fluorescent dyes and probes have been
developed as tools for high contrast microscopy and cell
imaging~\cite{1}. Ideal fluorescent probes should be bright,
photostable and non-toxic. Additional key parameters are their
absorption range and emission color. In terms of brightness,
fluorescent organic dyes may be surpassed by luminescent
nanoparticles~\cite{2,3}. Indeed, in the last decades, inorganic
nanoparticles have attracted growing attention in the field of
biophotonics, in relation with various applications including new
imaging modalities. Among them luminescent metal-, semiconductor- or
oxide-based nanoparticles have been by far the most widely
studied~\cite{4}. Luminescent metallic and semiconducting
nanoparticles have been heralded as major nanomaterials due to their
unique electronic and optical properties, giving rise to the blooming
field of plasmonics in the case of metallic nanoparticles. In parallel,
semiconductor quantum dots (QDs) have motivated many studies and have
become extremely popular for bioimaging uses. Their luminescence
properties can be tuned in the visible to the near-infrared (NIR)
region by adjusting their size in relation with quantum confinement and
they may show giant brightness~\cite{4} (both one-photon
brightness~\cite{3}: up to 
$10^{6}~\mathrm{M}^{-1}{\cdot}\mathrm{cm}^{-1}$, and two-photon
brightness~\cite{5,6,7,8}: up to $10^{4}$~GM). Yet, such
luminescent inorganic nanoparticles suffer from several drawbacks:
their inorganic core is inherently water-insoluble, requiring them to
be coated by solubilizing agents (polymeric coating, surfactants, etc.)
for use in bioimaging. In addition, several luminescent inorganic
nanoparticles raise questions with respect to environmental issues
(ecotoxicity, biodegradability) due to their content or composition
(heavy metal or toxic components). In that perspective, fully organic
nano-objects may offer interesting alternatives. The most widely used
typically are polymeric such as
dendrimers~\cite{9,10,11,12}, lipid-based~\cite{13} or
``frozen'' micelles systems where fluorescent probes are either
encapsulated in~\cite{14,15} or covalently linked~\cite{16} to
the nanoparticle. In contrast, self-stabilized organic nanoparticles
generated from the nano-aggregation of dedicated hydrophobic dyes in
water represent a much less traveled route. Actually, this type of
fully organic luminescent nanoparticles is often overlooked in the
literature and some prominent reviews do not even mention
them~\cite{3,17}. Yet they offer promise for a wide range of
applications, including biological applications~\cite{18a,18b}. We
propose to name these organic nanoparticles dFONs (for
\textit{dye-based fluorescent organic nanoparticles}) to distinguish
them from the multitude of fluorescent organic nanoparticles, which
otherwise create a somewhat fuzzy domain and some misunderstandings or
confusion. We stress that we use the name dFONs to describe
\textit{organic nanoparticles made only of pure dyes}, requiring
\textit{no stabilizers or additives, nor coatings} to be made
dispersible and stable in water or aqueous environments. 



The ultimate molecular confinement of pure dyes may look like a
counterintuitive approach as most of the time this generates
aggregation-caused quenching of fluorescence (ACQ) due to symmetry
reasons (in the case of $\uppi$-stacking notably) or to competitive
deactivation processes (such as photoinduced electron transfer (PET)
favored by the close proximity of dyes within dFONs) that inhibit
fluorescence. Yet by adjusting the electronic structure of the dye (to
prevent PET) while adding suitable bulky substituents that hinder
$\uppi$-stacking, such effects can be circumvented. A supposedly
similar strategy has been used extensively in the so-called
``aggregation-induced emission'' (AIE)~\cite{19} or
aggregation-induced emission enhancement (AIEE) domain. In compounds
designed for AIE, appending moieties preventing $\uppi$-stacking and
allowing for efficient non-radiative rotational deactivation processes
in non-viscous environments are added to dyes, thus generating loss of
fluorescence in solutions. When such dyes are placed in viscous
solvents or in the solid state where rotations are restricted, their
fluorescence is restored.

\section{dFONs design: polar and polarizable dyes (PPDs) as dFONs
building blocks}

Key criteria for luminescent nanoparticles to be of interest for
bioimaging purposes typically are large  brightness for enhanced
sensitivity (with the ultimate goal of detecting single emitters),
photostability, colloidal stability as well as biocompatibility.\unskip\break In  
addition, for {in vivo} bioimaging in tissues or small animals, they
should preferably absorb and emit light above 650~nm---i.e., within
the biological transparency window(s) (650--950~nm and
1000--1200~nm)~\cite{20}. In that perspective, two-photon excitation
has attracted much interest giving rise to two-photon laser scanning
microscopy (TPLSM)~\cite{21}, which has nowadays become a popular
fluorescence bioimaging technique. Two-photon absorption (2PA) is a
nonlinear optical phenomenon defined by the simultaneous absorption of
two low-energy photons~\cite{22} by an excitable compound
(Scheme~\ref{sch1}). The wavelength used for two-photon excitation is
thus favorably redshifted as compared to that used for one-photon
excitation and allows for instance the replacement of excitation in the
near UV--blue visible region by excitation in the deep red--NIR-I
region. Dyes which have much higher two-photon absorption
cross-sections ($\upsigma_{2}$) than endogenous chromophores (typically
orders of magnitude larger, i.e., over $10^{3}$~GM) are thus required
not only to allow deeper imaging in biological tissues thanks to
reduced scattering, but also to significantly reduce background
fluorescence and provide more selective excitation compared to standard
one-photon excitation~\cite{23}. In that perspective, we were
interested in the design of biocompatible dFONs combining tunable
emission, giant brightness (both for standard excitation, i.e.,
confocal fluorescence microscopy, and TPLSM), excellent colloidal and
photo-stability in bioenvironments, aiming at various imaging
modalities (including single particle tracking and two-photon imaging).


\begin{scheme*}
\includegraphics{sc01}
{\vspace*{6pt}}
\caption{\label{sch1}Two-photon excited fluorescence: principles and
advantages in bioimaging.}
{\vspace*{4pt}}
\end{scheme*}


Two main strategies are used to prepare dFONs directly as a colloidal
suspension in water (or aqueous media): (i) laser-assisted
photofragmentation of a suspension of micro-crystals in
water~\cite{24} or (ii)~nanoprecipitation~\cite{25,26}
(Scheme~\ref{sch2}). In the latter, a minute amount of a stock solution
of the dye dissolved in a water-miscible organic solvent (typically
THF, ethanol, acetone, DMSO\,\ldots)---which acts as the good solvent
for the hydrophobic dye---is added in a large volume of pure water (or
buffer etc.) under either magnetic stirring or sonication. For
bioimaging applications, no more than 1\% (v/v) of organic solvent
should be used and the residual amount can be eliminated by evaporation
under vacuum or dialysis. As the good (organic) solvent for the
hydrophobic dyes is dispersed in the bad solvent \mbox{(water),} dyes
aggregate to form clusters then nanoparticles~\cite{25}. If
nanoparticles are indeed formed, a colored (thanks to the dispersion of
hydrophobic dyes in water) and non-turbid (thanks to the nanometric
size of organic aggregates) aqueous suspension is obtained. We stress
that this process is a kinetically controlled step contrary to
crystallization and that hydrophobic interactions play a definite role
as water is used to conduct nanoprecipitation. Hence, the organization
of dyes within dFONs might be different from that in the crystal
obtained by a thermodynamically controlled slow process. dFONs prepared
by nanoprecipitation usually have a spherical shape. We emphasize that
the colloidal stability of such dFONs suspensions is a major issue in
view of their usefulness: their natural fate is to agglomerate, then
deposit upon evolution towards the low-energy thermodynamic state. The
nature of the dyes as well as their self-organization are extremely
important in that respect as the kinetics of these processes may be
rendered extremely slow depending on the type of dye and surface
properties of dFONs. \looseness=-1

\begin{scheme}
\vspace*{2pt}
\includegraphics{sc02}
{\vspace*{8pt}}
\caption{\label{sch2}Typical nanoprecipitation protocol for the
preparation of dFONs.}
\vspace*{-8pt}
\end{scheme}

To achieve dFONs that meet the requirements mentioned above for
bioimaging, we developed a successful bottom-up strategy based on the
molecular engineering of specific chromophores, \textit{namely Polar
and Polarizable Dyes (PPDs)} as dFONs building blocks. Such dyes bear
electron-releasing (D) and electron-withdrawing (A) moieties or
substituents that interact via $\uppi$-conjugated linkers, giving rise
to intramolecular charge transfer(s) (ICT), i.e., electronic
redistribution from the D to the A moieties upon excitation. The ICT
phenomenon is responsible for the strong absorption of PPDs as well as
for their unique nonlinear optical properties, especially two-photon
absorption~\cite{27,28,29,30}. PPDs may have different
symmetry including dipolar,  quadrupolar, or octupolar dyes
(Figures~\ref{fig1}--\ref{fig2})~\cite{31,32,33,34,35,36,37,38,39,40,41}.

\begin{figure*}
\includegraphics{fig01}
{\vspace*{2pt}}
\caption{\label{fig1}Examples of dipolar, octupolar and bis-dipolar
dyes developed
in the team for the bottom-up molecular engineering of
bright dFONs for bioimaging. Adapted from Refs.
\cite{31,32,33,34,37,39,41}.}
\end{figure*}


\begin{figure*}
\includegraphics{fig02}
\caption{\label{fig2}Examples of quadrupolar dyes developed in the team for the
bottom-up molecular engineering of bright dFONs for
bioimaging. Adapted from Refs.~\cite{35,36,38,39,40}.}
\vspace*{-2pt}
\end{figure*}





The presence of dipolar subunits in those dyes has strong implications
that explain the specificity and peculiarities of dFONs made from PPDs:
\begin{enumerate}[(i)]
\item The dipolar interactions between dyes are strong and longer-ranged
(${\propto} 1/r^{3}$) than Keesom interactions (${\propto} 1/r^{6}$) due
to the dyes' immobilization and ultimate confinement within dFONs.
These electrostatic interactions play a crucial role in the structural
cohesion of dFONs and explain their \textit{structural stability though
there is no covalent bonding between dyes}, contrary to polymeric
organic particles.
\item The dipolar interactions also play a major role in the
self-organization of PPD dyes within dFONs during the nano-aggregation
process~\cite{42,43}. In that respect, molecular engineering of
PPDs is crucial to prevent antiparallel stacking of dipolar dyes or
$\uppi$-staking of quadrupolar dyes~\cite{44} that are deleterious
to fluorescence. To prevent such processes, we have used dedicated
appending moieties such as diphenylamino end-groups (which have a
non-planar propeller shape and are also electron-releasing moieties),
as in dipolar and quadrupolar dyes shown in Figures~\ref{fig1}
and~\ref{fig2}. Alternatively, conjugated linkers (or cores) including
fluorenyl moieties bearing alkyl substituents that\unskip\break 
extend above and
below the plane of the $\uppi$-conjugated system have been used. These
substituents act as bulky spacers of tunable size, as in bis-dipolar
and quadrupolar dyes shown in Figures~\ref{fig1} and~\ref{fig2}. The
geometry of the dyes, as well as the position and size of the bulky
appending moieties, are very important in tuning their
self-organization upon aggregation and offer a powerful\unskip\break handle for
bottom-up engineering of the dFONs' fluorescence properties
(Figure~\ref{fig3}). 
\item The dipolar subunits create strong local electric fields in their
close vicinity. As a result, due to the intrinsic high polarizability
of PPDs, these local electric fields may change the polarization of the
proximal dyes resulting in a modification of their electronic
distribution (and polarization) and subsequently of their optical
properties (i.e., modification of the electronic gap
\cite{45,46,47,48,49,50}) and even more markedly
nonlinear optical properties~\cite{29,51,52}. Hence, depending
on the self-orientation of the dipolar subunits within dFONs and on the
distance between PPDs dyes (which can be tuned by adjusting the
geometry of the dyes as well as the \mbox{bulkiness} and position of
substituents---Figure~\ref{fig3}), sizeable variations in the optical
properties can be expected for dyes with similar optical (and NLO)
properties in solution as a result of confinement effects.
\item Finally, the nature of the PPDs may significantly influence their
orientation at the surface of dFONs resulting in short-range
order~\cite{53}. We stress that almost all push--pull dyes that we
investigated bear electron-withdrawing end-groups that are susceptible
to favor electrostatic interaction or H-bonds with water molecules
surrounding the surface of the dFONs. Such orientations may result in
large surface potentials (as in lipid membranes), emphasizing the
specificity of dFONs made from PPDs in terms of the nature and
properties of their surface.
\end{enumerate}

\begin{figure*}
\includegraphics{fig03}
\caption{\label{fig3}Tuning the organization of dipolar (arrow) and
quadrupolar (double arrow) dye subunits in dFONs and its impact
on fluorescence intensity.}
\end{figure*}


\section{Bottom-up engineering of optical properties}

In view of optical bioimaging, ``redder is better'' due to improved
penetration depth and easier detection. In that perspective, we have
been keen on red-shifting the absorption and emission of dFONs
(bathochromic shift) and increasing their one- and two-photon response
(hyperchromic effect) as well as their one- and two-photon
brightnesses.


\subsection{Adjusting the strength of D and A moieties} 

A classical strategy involves the increase in the D/A strength or the
extension of the $\uppi$-linker. Such a strategy is illustrated in
Figure~\ref{fig4}A for dipolar dyes. We note that a bathochromic shift
is observed both in solution and in dFONs~when increasing the strength
of the donor or acceptor. A hyperchromic shift is also observed, such
that for dFONs of similar size (i.e., same amount of dye subunits), the
ones with a stronger donor or acceptor have a larger absorptivity. A
bathochromic shift of the emission is also observed such that
\textit{dFONs made from dipolar dyes} $\mathbf{D}_{\mathbf{D3}\boldsymbol{\uppi}
\mathbf{2A1}}$ \textit{and} $\mathbf{D}_{\mathbf{D3}
\boldsymbol{\uppi}\mathbf{2A5}}$ \textit{are respectively red--orange and NIR-I
emitters that show huge brightnesses in water, larger than those of} QDs
\textit{without the need for surfactants or coating}. We also note the effect
of the size of substituents on fluorescence intensity: dFONs made from
$\mathbf{D}_{\mathbf{D3}\boldsymbol{\uppi} \mathbf{2A1}}$ have an almost twice
larger quantum yield than dFONs made from $\mathbf{D}_{\mathbf{D1}\boldsymbol{\uppi}
\mathbf{2A1}}$, emphasizing the role of substituent size on the
organization of PPDs within dFONs\break (Figure~\ref{fig4}A). 



\begin{figure*}
\includegraphics{fig04}
\caption{\label{fig4}Effect of donor and acceptor end-group strength and
$\pi$-linker length on the optical properties of dFONs made
from dipolar (``push--pull'') dye (A: top) and quadrupolar dyes (B:
bottom). Adapted from Refs.~\cite{31,33,34} and~\cite{39,40}.}
\end{figure*}





Subtle tuning can be achieved by tweaking the donor strength or the
acceptor strength both for dipolar~\cite{32,39} and quadrupolar
dyes~\cite{40} (Figure~\ref{fig4}B). 


\subsection{Adjusting the $\uppi$-linker's nature and length}\label{sec3.2}

Similarly, a hyperchromic and bathochromic shift can be achieved by
adjusting the length and nature of the $\uppi$-linkers as illustrated
in Figure~\ref{fig4}A for dipolar dyes and in Figure~\ref{fig5} for
quadrupolar dyes~\cite{36,39}.

\begin{figure*}
\includegraphics{fig05}
\caption{\label{fig5}Effect of $\uppi$-linker length and structure on the optical
properties of dFONs made from quadrupolar dyes. Adapted from Ref.
\cite{36}.}
\end{figure*}


We note that similar trends are observed for dyes in solution and
dFONs. On the other hand, the observed changes in fluorescence quantum
yield emphasize the impact of dye size and geometry on dye packing
within dFONs in the case of \mbox{quadrupolar} dyes. Indeed dFONs made from
$\mathbf{Q1}_{\mathbf{A2}\boldsymbol{\uppi}\mathbf{3R1D1}}$ show a much
lower fluorescence quantum yield than dFONs made from
$\mathbf{Q1}_{\mathbf{A2}\boldsymbol{\uppi}\mathbf{1R1D1}}$ and
$\mathbf{Q1}_{\mathbf{A2}\boldsymbol{\uppi}\mathbf{2R1D1}}$ and even than
dFONs made from $\mathbf{Q1}_{\mathbf{A2}\boldsymbol{\uppi}\mathbf{4R1D1}}$
although the latter are significantly redshifted (Figure~\ref{fig5}).
As a result, \textit{dFONs made from quadrupolar dyes}
$\mathbf{Q1}_{\mathbf{A2}\boldsymbol{\uppi}\mathbf{1R1D1}}$ \textit{and}
$\mathbf{Q1}_{\mathbf{A2}\boldsymbol{\uppi}\mathbf{4R1D1}}$ \textit{are respectively
orange--red and deep red emitters that exhibit a very large\break
brightness}.


\subsection{Adjusting the geometry of the PPDs and the size of
substituents to tune packing and fluorescence properties} 
\label{sec3.3}

Not only may the geometry of the PPDs significantly influence the
fluorescence quantum yield (and thus affect the brightness) of dFONs,
but it can also be used to change the emission color by controlling and
exploiting the local environment created by local dipoles within dFONs.
Indeed, whereas dipolar $\mathbf{D}_{\mathbf{A1}\boldsymbol{\uppi}\mathbf{1D1}}$ and
related octupolar $\mathbf{O}_{\mathbf{A1}\boldsymbol{\uppi}\mathbf{1D4}}$ PPDs have
identical fluorescence properties in solution (fluorescence quantum
yields and lifetimes) and show the same emission solvatochromism (blue
emission in toluene, green emission in chloroform and orange emission
in DMSO), dFONs made from $\mathbf{D}_{\mathbf{A1}\boldsymbol{\uppi}\mathbf{1D1}}$
are green emitters whereas dFONs made from
$\mathbf{O}_{\mathbf{A1}\boldsymbol{\uppi}\mathbf{1D4}}$ are orange emitters: the
three-branched geometry of $\mathbf{O}_{\mathbf{A1}\boldsymbol{\uppi}\mathbf{1D4}}$
imposes a different packing from the rod-like
$\mathbf{D}_{\mathbf{A1}\boldsymbol{\uppi}\mathbf{1D1}}$, generating a stronger
local electric field such that
$\mathbf{O}_{\mathbf{A1}\boldsymbol{\uppi}\mathbf{1D4}}$ dyes experience a larger
polarity within dFONs (similar to DMSO) and its emission is
redshifted~\cite{31}.


Another way to influence the packing of PPDs and tune their
luminescence properties is to adjust the size of the bulky
substituents. A striking example is given in Figure~\ref{fig6} for a
series of quadrupolar dyes built from a fluorenyl core. All dyes bear
the same electron-withdrawing end-groups and $\uppi$-linker, the only
difference being the nature of the substituents; cyclopentyl,
cyclobutyl and cyclopropyl for dyes with a spirofluorene core or two
\textit{iso}pentyl chains for dyes with a bis-alkyl fluorene core.
Whereas all quadrupolar dyes show identical optical properties in
solution, dFONs fluorescence is strongly affected~\cite{54}.
Decreasing the size of the cycle in spirofluorenyl derivatives
generates a redshift of the dFONs fluorescence, allowing one to tune
the dFONs fluorescence from blue (cyclopentyl), to cyan (cyclobutyl)
and to red--orange (cyclopropyl). This illustrates the effect of subtle
tuning of the side-chain size on the packing of the dyes and thus on
fluorescence. We note that the quadrupole with two \textit{iso}pentyl
chains leads to dFONs whose fluorescence is both redshifted (green
emission) compared to its spiro (cyclopentyl) analog and enhanced by a
factor of 2, demonstrating the effect of substituent size and bulkiness
on dye packing and its consequences on optical\break properties.

\begin{figure*}
\includegraphics{fig06}
\vspace*{-3pt}
\caption{\label{fig6}Influence of the substituents on the fluorenyl core of
quadrupolar dyes on the fluorescence emission of corresponding dFONs in
water.}
\vspace*{-2pt}
\end{figure*}


\subsection{Enhancing two-photon absorption. Effects of confinement} 

The two-photon absorption of dipolar and quadrupolar dyes can be
modulated by molecular engineering, adjusting the donor and acceptor\unskip\break
groups as well as the length and nature of the $\uppi$-conjugated
linker~\cite{27,28,30,51}. Increasing the strength of the donor or
acceptor groups in dipolar thienothiophene~\cite{32} or
bisthienyl~\cite{39} dyes allows one to significantly increase their
response in solution as illustrated in Figure~\ref{fig7}A. Yet, the
molecular confinement of dipolar dyes within dFONs has a major effect
on their 2PA response; either a major decrease (by a factor of 3 or 2
for $\mathbf{D}_{\mathbf{A4}\boldsymbol{\uppi}\mathbf{3D1}}$ and
$\mathbf{D}_{\mathbf{A4}\boldsymbol{\uppi}\mathbf{3D3}}$) or an increase
($\mathbf{D}_{\mathbf{A1}\boldsymbol{\uppi}\mathbf{3D1}}$ and
$\mathbf{D}_{\mathbf{A4}\boldsymbol{\uppi}\mathbf{3D2}}$) of the 2PA cross-section
of the dipolar dyes is observed upon confinement within dFONs
(Figure~\ref{fig7}A). This phenomenon can be ascribed to electrostatic
interactions between dipolar and polarizable dyes depending on their
relative orientation and proximity~\cite{11,29}. The striking
difference between the 2PA response of dyes
$\mathbf{D}_{\mathbf{A4}\boldsymbol{\uppi}\mathbf{3D1}}$ and
$\mathbf{D}_{\mathbf{A4}\boldsymbol{\uppi}\mathbf{3D2}}$ into dFONs illustrates the
positive role of the Br substituent in tuning the packing and allowing
a cooperative enhancement of the 2PA~\cite{11}, as is also the case for
$\mathbf{D}_{\mathbf{A1}\boldsymbol{\uppi}\mathbf{3D1}}$ (Figure~\ref{fig7}A).
Although the fluorescence quantum yields of NIR-I-emitting dFONs made
from dipolar dyes $\mathbf{D}_{\mathbf{A4}\boldsymbol{\uppi}\mathbf{3D2}}$ and
$\mathbf{D}_{\mathbf{A4}\boldsymbol{\uppi}\mathbf{3D3}}$ remain low in water, their
large 2PA response and number of dye subunits in dFONs lead to a very
large two-photon brightness at 1070~nm (Figure~\ref{fig7}A). As such,
they provide interesting nanoprobes for NIR-II to NIR-I bioimaging in
living tissues.

\begin{figure*}
\vspace*{-3pt}
\includegraphics[scale=0.3]{Fig7-1.png}
%\vspace*{-1.3pc}
\caption{\label{fig7}Molecular engineering of the two-photon absorption
of dFONs made from dipolar~\cite{32} (A: top) or quadrupolar~\cite{36}
(B: bottom) dyes.}
\end{figure*}


\begin{figure*}
\vspace*{-1pt}
\includegraphics{fig08}
\caption{\label{fig8}Molecular engineering of the colloidal and structural
stability of dFONs made from dipolar dyes. Adapted from Refs.~\cite{32}
and \cite{34}.}
\vspace*{-1pt}
\end{figure*}


The increase in length leads to a redshift of the 2PA as was the case
for the 1PA, but it also induces a major increase in the two-photon
absorption response of the quadrupolar dyes, both in solution and as
dFONs building blocks. (Figure~\ref{fig7}B). The nature of the
elongating moieties (ethynyl, vinyl or thienyl) influences not only the
fluorescence quantum yield and the emission color (see
Section~\ref{sec3.2}) but also the 2PA response: an enhancement is
observed upon increased conjugation~\cite{36}. Interestingly, in all
cases, confinement does not reduce the 2PA response of the dyes but
rather leads to broadening of the 2PA spectrum~\cite{36}. Thanks to the
combined variation of the 2PA responses and of the fluorescence quantum
yields values, the orange--red and deep red emitting dFONs made from
$\mathbf{Q1}_{\mathbf{A2}\boldsymbol{\uppi}\mathbf{1R1D1}}$ and 
$\mathbf{Q1}_{\mathbf{A2}\boldsymbol{\uppi}\mathbf{4R1D1}}$ also show the largest 2P
brightness. As such, they represent bright nanoprobes for NIR-I to red
bioimaging~\cite{36}.


\subsection{Enhancing brightness by increasing size} 

The brightness of dFONs made from PPDs can be enhanced simply by
increasing the size of the dFONs, as is the case for QDs. This can be
achieved by adjusting the nanoprecipitation conditions, notably the dye
concentration of the stock solution in the organic solvent~\cite{26}.
Contrarily to QDs, this does not lead to a change of the luminescence
properties (emission spectrum, fluorescence quantum yield) as shown in
the case of dFONs made from dipolar dye
$\mathbf{D}_{\mathbf{A1}\boldsymbol{\uppi}\mathbf{2D1}}$~\cite{34} and quadrupolar
dye $\mathbf{Q1}_{\mathbf{A2}\boldsymbol{\uppi}\mathbf{1D1}}$~\cite{35}. Hence, dFONs
with a similar emission color but different sizes (and thus
brightnesses) can be obtained from PPDs, as can dFONs with a similar
size but different emission colors~\cite{31,34,36}.


\section{Bottom-up engineering of surface properties}
\subsection{Molecular engineering of dFONs stability}

Strikingly, molecular engineering of the PPDs also allows tuning of the
surface properties of dFONs. dFONs made from PPDs show good colloidal
\mbox{stability} in relation with large surface potentials. This kinetic
stability can be further enhanced by adjusting the structure of the
PPDs. As illustrated in Figure~\ref{fig8}, increasing the size of the
$\uppi$-conjugated linker~\cite{34} or adding bulky substituents on the
donor end-group~\cite{32} or the nature of the acceptor
end-group~\cite{33} of dipolar dyes allow increasing the colloidal and
structural stability of dFONs. The evolution of the fluorescence
quantum yield values over time is of major importance for the usability
of dFONs for bioimaging but also the signature of the structural
stability of dFONs made from dipolar dyes: the decrease in fluorescence
over time is related to the exchange of dyes from the surface to the
bulk water back to the surface, leading to surface defects that are
responsible for fluorescence quenching. Hence, the larger the
interaction energy of the dyes with the surface (i.e., the energy
required to extract a surface dye from the surface to the bulk), the
slower the process~\cite{53}. Therefore stronger electrostatic
interchromophoric interactions within dFONs and addition of hydrophobic
substituents allow improving their colloidal and structural stability.
As a result, dFONs made from 
$\mathbf{D}_{\mathbf{A5}\boldsymbol{\uppi}\mathbf{2D3}}$ show very high colloidal
and structural stability~\cite{33}.

\subsection{Molecular engineering of dFONs cell internalization and
stealthiness} \label{sec4.2}

Strikingly, the interaction of dFONs with cell membranes can also be
``bottom-up-engineered'' by adjusting the structure of the PPDs. dFONs
made from the dipolar dyes $\mathbf{D}_{\mathbf{A5}\boldsymbol{\uppi}\mathbf{2D3}}$
were found to be non-toxic and to internalize within cells while
retaining their structural integrity~\cite{33}. In contrast, articulated
dipoles ($\mathbf{Ad}_{\mathbf{A1D5}\boldsymbol{\uppi} \mathbf{5R}}$,
Figure~\ref{fig1}) bearing two long hydrophobic chains were found to
show no non-specific interactions with cell membranes, exhibiting an
intrinsically stealth behavior~\cite{41}. Hence, such \textit{dFONs made
only of pure PPDs} do not require specific coatings (PEG or
zwitterionic) to be made stealth. As such, thanks to their high
brightness and intrinsic stealth character, they hold promise for
imaging of specific receptors in cells once surface-functionalized with
specific targeting moieties. 

Similarly, dFONs made from quadrupolar dyes built from a spiroflurenyl
core ($\mathbf{Q}_{\mathbf{2CR3}\boldsymbol{\uppi}\mathbf{5D1}}$ and
$\mathbf{Q}_{\mathbf{2CR3}\boldsymbol{\uppi}\mathbf{4A4}}$) were found to
internalize within cells~\cite{38} while retaining their structural
{integrity.} In contrast, quadrupoles
$\mathbf{Q1}_{\mathbf{A2}\boldsymbol{\uppi}\mathbf{1R1D1}}$~\cite{35},
$\mathbf{Q1}_{\mathbf{A2}\boldsymbol{\uppi}\mathbf{2R1D1}}$~\cite{36},
$\mathbf{Q1}_{\mathbf{A2}\boldsymbol{\uppi}\mathbf{2R1D2}}$~\cite{40},
$\mathbf{Q1}_{\mathbf{A2}\boldsymbol{\uppi}\mathbf{21RD3}}$~\cite{40} and
$\mathbf{Q1}_{\mathbf{A2}\boldsymbol{\uppi}\mathbf{4R1D1}}$~\cite{36}, with a rigid
elongated \mbox{conjugated} system bearing four hydrophobic alkyl chains were
found to be non-toxic and to exhibit a stealth behavior. Interestingly
$\mathbf{Q1}_{\mathbf{A2}\boldsymbol{\uppi}\mathbf{3R1D1}}$ does not show a stealth
behavior indicating that both the presence of several lipophilic chains
and the nature of the $\uppi$-conjugated system are decisive in
controlling the nano-bio interface~\cite{36,40}.

\begin{figure*}
\vspace*{3pt}
\includegraphics{fig09}
\vspace*{3pt}
\caption{\label{fig9}Two-photon angiography in small animals.
Left, 3D projection Two-Photon Excitation Fluorescence (TPEF)
and SHG ($\uplambda^{\mathrm{2P}}$ @820~nm) image 90~min after injection of
dFONs made from $\mathbf{D}_{\mathbf{A1}\boldsymbol{\uppi}\mathbf{1D1}}$. Right, 3D
projection TPEF ($\uplambda^{\mathrm{2P}}$ @820~nm) image 90~min after
injection of dFONs made from
$\mathbf{O}_{\mathbf{A1}\boldsymbol{\uppi}\mathbf{1D4}}$. For all images, the red
color corresponds to the endogenous SHG signal (second harmonic
generation at 410~nm) that arises from muscle tissue, while the green
corresponds to the TPEF signal generated {in vivo} by the dye 
(460--590~nm). Scale bar: $100~\upmu\mathrm{m}$. Adapted from
Ref.~\cite{31}.}
\vspace*{2pt}
\end{figure*}

Furthermore, thanks to their highly negative surface potential, these
dFONs can be surface-coated by a polycationic polymer, resulting in a
reversal of the surface potential. This change in surface potential
triggers the internalization of the dFONs within cells, opening a
promising route towards innovative drug delivery
protocols~\cite{36}.

\section{Bioimaging applications ({in vitro} and {in vivo})}

The unique combination of high colloidal stability, small size, giant
brightness and tunability of dFONs made from PPDs makes them ideal
candidates for bioimaging in dilute conditions (i.e., nM range).


\begin{figure*}
\vspace*{-4pt}
\includegraphics{fig10}
\vspace*{-3pt}
\caption{\label{fig10}\fontsize{9.93}{12}\selectfont Single-particle
tracking of dFONs made from
$\mathbf{D}_{\mathbf{A5}\boldsymbol{\uppi}\mathbf{2D3}}$,
$\mathbf{Ad}_{\mathbf{A1D5}\boldsymbol{\uppi}\mathbf{5R1}}$ and
$\mathbf{Ad}_{\mathbf{A1D5}\boldsymbol{\uppi}\mathbf{5R3}}$ in water.
Single-particle tracking of dFONs made from
$\mathbf{D}_{\mathbf{A5}\boldsymbol{\uppi}\mathbf{2D3}}$ (left),
$\mathbf{Ad}_{\mathbf{A1}\boldsymbol{\uppi}\mathbf{5D5R1}}$ (middle) in water and
$\mathbf{Ad}_{\mathbf{A1}\boldsymbol{\uppi}\mathbf{5D5R3}}$ (right) in phosphate
buffer saline (with 10\% of fetal bovine serum). Examples of traces of
single particles with corresponding histograms of hydrodynamic diameter
are given. Adapted from Refs.~\cite{33} and \cite{41}.}
\vspace*{-3pt}
\end{figure*}


\subsection{Two-photon angiography in small living animals~\cite{31}}

As discussed earlier, the molecular design of PPDs is crucial for dFONs
properties with consequences on bioimaging applications. A typical case
is the difference between dFONs made from dipolar dye
$\mathbf{D}_{\mathbf{A1}\boldsymbol{\uppi}\mathbf{1D1}}$ and from its three-branched
(octupolar) version $\mathbf{O}_{\mathbf{A1}\boldsymbol{\uppi}\mathbf{1D4}}$ when
used as a contrast agent for angiography~\cite{31}. Both dFONs show
a very large two-photon brightness ($10^{6}$ and $2 \times 10^{5}$~GM
respectively for dFONs of about 35~nm in diameter)~\cite{31}, one being
a green emitter and the other an orange emitter. These dFONs were
injected in the blood circulation of living \textit{Xenopus laevis}
tadpoles. The tail of the tadpole was then imaged by
two-photon-excited\unskip\break fluorescence (TPEF) 20~min and 90~min after
injection. Both dFONs allow the high contrast and high resolution
visualization of blood vessels in the tiny animal (Figure~\ref{fig9}) by
two-photon excitation in the NIR region (820~nm) and detection of the
green or red--orange fluorescence emitted by dFONs made from
$\mathbf{D}_{\mathbf{A1}\boldsymbol{\uppi}\mathbf{1D1}}$ or
$\mathbf{O}_{\mathbf{A1}\boldsymbol{\uppi}\mathbf{1D4}}$ dyes. Thanks to their
nanoscale, the dFONs do not permeate through the walls of the blood
vessels, thus allowing their imaging with high resolution. Furthermore,
no toxicity is observed for the dFONs made from
$\mathbf{D}_{\mathbf{A1}\boldsymbol{\uppi}\mathbf{1D1}}$. Yet, this is no longer
true for the dFONs made from
$\mathbf{O}_{\mathbf{A1}\boldsymbol{\uppi}\mathbf{1D4}}$, which quickly agglomerate
into large micro-agglomerates resulting in a deadly clogging of the
blood vessels. Hence, not only does the geometry of the dye influence
the optical properties of dFONs (see Section~\ref{sec3.3}), but it also
dramatically impacts the nature of their nano--bio interface and
consequently their usability as a contrast agent for angiography\break
by TPEF. 

\begin{figure*}
{\vspace*{-1pt}}
\includegraphics{fig11}
{\vspace*{-1pt}}
\caption{\label{fig11}Simultaneous bicolor single-particle tracking within COS cells.
Simultaneous bicolor single-particle tracking of dFONs made
from $\mathbf{Q2}_{\mathbf{CR3}\boldsymbol{\uppi}\mathbf{4A4}}$ and
$\mathbf{Q2}_{\mathbf{CR3}\boldsymbol{\uppi}\mathbf{5D1}}$ in live COS7 cells; A. wide
field, COS7 cell contour is drawn in black; B. Merged single particle
tracks on green and red channels; C. Live tracking of
$\mathbf{Q2}_{\mathbf{CR3}\boldsymbol{\uppi}\mathbf{4A4}}$  dFONs (boxes) in COS7
cells on red channel; D. Live tracking of
$\mathbf{Q2}_{\mathbf{CR3}\boldsymbol{\uppi}\mathbf{5D1}}$ dFONs (boxes) in COS7 cells
on green channel; Scale bar $=$~$5~\upmu\mathrm{m}$, excitation at 488~nm
($P = 306~\mathrm{W}{\cdot}\mathrm{cm}^{-2}$). Adapted from
Ref.~\cite{38}.}
{\vspace*{-1pt}}
\end{figure*}

\subsection{Single-particle tracking in solutions, cells and in the brain}

Single-particle tracking (SPT) is a class of techniques that allow one
to dynamically follow the fate of single molecules in living tissues.
In this respect, QDs are the most widely used nanoparticles thanks to
their unprecedented brightness and photostability. Due to the giant
brightness of dFONs made from PPDs (similar or higher than those of
QDs), they represent promising candidates for SPT and their usability
for SPT in aqueous solutions was investigated. A critical parameter in
that respect is their photostability. Organics are recognized to be
much less photostable than inorganics. Yet depending on the structure
of the PPDs, we found that the brightness of dFONs made from dipolar
($\mathbf{D}_{\mathbf{A5}\boldsymbol{\uppi}\mathbf{2D3}}$) or quadrupolar
($\mathbf{Q}_{\mathbf{2CR3}\boldsymbol{\uppi}\mathbf{5D1}}$ and
$\mathbf{Q}_{\mathbf{2CR3}\boldsymbol{\uppi}\mathbf{4A4}}$) dyes allows performing
single particle tracking with adequate~\cite{33}, good~\cite{38} to
excellent photostability (up to 3~min)~\cite{38}. The reconstruction of
the trajectories of single particles gives access to the hydrodynamic
diameter of the nanoparticles. The hydrodynamic size of dFONs made from
articulated dipole-based
($\mathbf{Ad}_{\mathbf{A1D5}\boldsymbol{\uppi}\mathbf{5R1}}$, 
$\mathbf{Ad}_{\mathbf{A1D5}\boldsymbol{\uppi}\mathbf{5R2}}$ and 
$\mathbf{Ad}_{\mathbf{A1D5}\boldsymbol{\uppi}\mathbf{5R3}}$) was found to be similar
to the dry diameter measured by transmission electronic
microscopy~\cite{41}. Interestingly, SPT experiments conducted in
saline aqueous buffer containing proteins (i.e., PBS with fetal bovine
serum) reveal that the hydrodynamic size of articulated dipole-based
($\mathbf{Ad}_{\mathbf{A1D5}\boldsymbol{\uppi}\mathbf{5R1}}$, 
$\mathbf{Ad}_{\mathbf{A1D5}\boldsymbol{\uppi}\mathbf{5R2}}$ and 
$\mathbf{Ad}_{\mathbf{A1D5}\boldsymbol{\uppi}\mathbf{5R3}}$) dFONs
increases by ${\sim}$30--40\% (Figure~\ref{fig10}) as compared to that
determined in pure
water~\cite{41}. Such an increase reveals the formation of a protein
corona at the surface of the nanoparticles, thus preventing aggregation
of dFONs despite the ionic strength of the solution. The presence of
such a corona is definitely an advantage for bioimaging in live cells
when dFONs are used as tracers.

\begin{figure*}
\includegraphics{fig12}
\caption{\label{fig12}Single-particle tracking deep into brain tissues.
Single-particle tracking of small (10~nm) dFONs made from
$\mathbf{Q1}_{\mathbf{A2}\boldsymbol{\uppi}\mathbf{1D1}}$ in organotypic brain
slices. (A) Organotypic brain slices preparation after extraction of
the mouse brain and dissection of the hippocampus. (B) Trace of
single-particle tracking for 11~s, $150~\upmu\mathrm{m}$ below the
surface of an organotypic brain slice. (C) Fluorescence images of
single QDs-655 and $\mathbf{Q1}_{\mathbf{A2}\boldsymbol{\uppi}\mathbf{1D1}}$
dFONs recorded at the indicated depths (an intensity profile of single
particle is provided on the right). Fields of view are 
$6.5~\upmu\mathrm{m}$ wide. Adapted from Ref.~\cite{35}.}
\end{figure*}

SPT experiments can also be performed with quadrupolar dye-based
($\mathbf{Q}_{\mathbf{2CR3}\boldsymbol{\uppi}\mathbf{5D1}}$ and
$\mathbf{Q}_{\mathbf{2CR3}\boldsymbol{\uppi}\mathbf{4A4}}$) dFONs \textit{within
cells}. Interestingly, due to their different sizes (43~nm and 14~nm
for $\mathbf{Q}_{\mathbf{2CR3}\boldsymbol{\uppi}\mathbf{5D1}}$ and
$\mathbf{Q}_{\mathbf{2CR3}\boldsymbol{\uppi}\mathbf{4A4}}$, respectively) and
different absorption spectra ($\uplambda_{\mathrm{abs}}^{\max} =
400$~nm and 503~nm for $\mathbf{Q}_{\mathbf{2CR3}\boldsymbol{\uppi}\mathbf{5D1}}$
and $\mathbf{Q}_{\mathbf{2CR3}\boldsymbol{\uppi}\mathbf{4A4}}$, respectively), the
two dFONs show similar and large brightnesses at 488~nm (wavelength of
the blue laser). Yet their emission can be discriminated easily as
dFONs made from $\mathbf{Q}_{\mathbf{2CR3}\boldsymbol{\uppi}\mathbf{5D1}}$ are green
emitters while dFONs made from
$\mathbf{Q}_{\mathbf{2CR3}\boldsymbol{\uppi}\mathbf{4A4}}$ are NIR-I emitters.
Thanks to these unique properties, these dFONs that internalize into
cells (see Section~\ref{sec4.2}) can be tracked at the single-particle
level and used together for \textit{simultaneous bicolor SPT within
cells} (Figure~\ref{fig11})~\cite{38}.




To date, the wealth of information provided by SPT was mostly obtained
in simplified systems such as cell monolayers. However, the
microenvironment of a cell highly regulates its behavior and its
molecular dynamics. There is therefore a need to perform SPT deep in
intact tissues. Using small (10 nm in diameter), bright red-emitting
and stealth dFONs made from quadrupolar dyes
$\mathbf{Q1}_{\mathbf{A2}\boldsymbol{\uppi}\mathbf{1R1D1}}$, it was possible to
explore the extracellular brain space deeper into tissues than with the
equivalent QDs (i.e., QD655). The small dFONs diffuse deep into
organotypic brain slices and can be tracked down to 
$150~\upmu\mathrm{m}$ depth while QD655 are no longer
detected~\cite{35} (Figure~\ref{fig12}). 



\section{Conclusion and perspectives}

The molecular engineering of dedicated polar (``push--pull'') and
polarizable dyes as dFONs building blocks has proven to be a successful
approach to achieve ultrabright small luminescent organic nanoparticles
that compete with QDs for \mbox{bioimaging} purposes. Not only can their
optical properties be modulated, but their (photo)stability and surface
properties can be tuned by adjusting the structure of the PPDs. This
demonstrates that organics can also play an important role in the field  
of {Nanobiotechnologies,} opening new routes for nanotools for drug
delivery or super-resolution imaging. dFONs made from PPDs is a highly
versatile approach benefiting from the wide wealth of organic dyes.
Compared to inorganics, such dFONs may provide a safer and more
sustainable alternative: their toxicity can be tuned by adjusting the
structure of the dyes, while their synthesis can benefit from green
protocols. \looseness=-1

An important aspect is their surface functionalization for applications
such as early diagnostic, ultrasensitive sensing and super-resolution
imaging. In that respect, two main approaches are developed:
non-covalent (electrostatic-based) functionalization~\cite{36} and
covalent functionalization~\cite{55}. We are currently investigating
these routes.


\section*{Declaration of interests}
The authors do not work for, advise, own shares in, or receive funds
from any organization that could \mbox{benefit} from this article, and have
declared no affiliations other than their research organizations.

\section*{Funding}

The Conseil general d'Aquitaine is acknowledged for financial support
(Chaire d'Excellence Grant to MBD). Financial supports for ODP and EK
is provided through the MESRI via the University of Bordeaux.
Continuous support from Univ. Bordeaux, CNRS and Bordeaux-INP is
acknowledged. This work was also supported by the ERC COMET
(101077364).

\CDRGrant[ERC COMET]{101077364}

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