Dr Suzanne Fery-Forgues

Davide

Sergio Slava

Laura

Valentina Cristiano

Silvia Luca

Dr Rainer Kling

Prof Wim

Prof Jean-Christophe Baret

Jessica Victor

Jeanne Thank

Corrado, Fajo, Marta, Margherita, Dano, Bigi Veronica Cami

Keywords: Two-photon absorption, fluorophores, organic nanoparticles, bioimaging UMR 5255

Look wide, and even when you think you are looking wide, look wider still

Elaboration et caractérisation de nanoparticules ultra-brillantes, fonctionnalisées et biocompatibles, pour applications en biologie et en médicine

L'utilisation de nano-objets luminescents en milieu biologique est devenue très répandue, notamment en vue d'applications biomédical est elles que l'imagerie, la thérapie et le diagnostic. Jusqu'à récemment, les principaux travaux réalisés dans ce domaine concernaient les nanoparticules de silice dopées ou fonctionnalisées avec des molécules organiques, les nanoparticules d'or et les nanoparticules semi-conductrices (quantum dots, i.e., QDs). Toutefois, un certain nombre de limitations demeurent pour les applications dans le domaine du vivant, en lien notamment avec des problèmes de stabilité, de biocompatibilité et de toxicité ou encore de biodégradabilité. En parallèle, un certain nombre de molécules organiques fluorescentes non-toxiques ont été utilisées comme sondes fluorescentes en milieu biologique, mais leur brillance demeure limitée. L'idée directrice de la thèse est de concevoir et synthétiser de nouveaux chromophores organiques présentant une émission modulable (du visible au proche infrarouge) et adaptés à la préparation de nanoparticules organiques fluorescentes (FONs) combinant à la fois une brillance extrêmement élevée, une excellente stabilité colloïdale et une photostabilité adaptée à leur utilisation en imagerie in vitro et in vivo. De tels nanoobjets ultra-brillants pourraient alors représenter une alternative très intéressante aux nanoparticules actuellement les plus utilisées en imagerie de fluorescence du vivant (QDs). Le manuscrit décrit la synthèse et les propriétés de plusieurs classes de molécules fluorescentes spécifiquement conçues pour former des telles FONS par auto-assemblage dans l'eau. La préparation de ces FONs est présentée et leurs propriétés étudiées et discutées. Enfin des applications concrètes en bio-imagerie sont présentées.

Mots clés: Absorption à deux photons, fluorophores, nanoparticules organiques, imagerie biologique

Design and Synthesis of ultra-bright Organic Nanoparticles (ONPs) for bioimaging

Nowadays the use of bright luminescent nano-objects in biological environment is a topic that is gaining more and more importance, especially for biomedical applications such as imaging, therapy and diagnostic. So far, numerous studies have been conducted with gold nanoparticles, silica nanoparticles (doped or functionalized with organic molecules), as well as semiconductor nanoparticles (quantum dots, i.e., QDs). However, most of these nanoparticles suffer from drawbacks (in terms of stability, biocompatibility, eco-toxicity or degradability). On the other hand, several nontoxic fluorescent molecular probes have been widely used, but most of the time their brightness remain modest in biological environments compared to QDs. Our idea is to engineer new organic chromophores with tunable emission wavelength (from visible to near infrared) for further preparation of organic fluorescent nanoparticles (so called FONs) that display giant one-photon and two-photon brightness, as well as good colloidal and chemical stability, and suitable photostability for in vitro and in vivo imaging. As such, these FONs would represent interesting alternatives to QDs for use in bioimaging. This manuscript describes the synthesis and characterization of new classes of fluorescent molecules specifically engineered as building blocks for the fast preparation of such nanoparticles by self-aggregation in water. The FONs were fully characterized from both morphological and photophysical points of view and further used in bioimaging.

TABLE OF CONTENT

Absorption of ultraviolet and visible light

The absorption of one photon by a molecule induces an electronic transition from a lower electronic state (typically the ground state) to higher electronic states called excited states. Depending on the molecular structure, the absorption can promote electrons from σ, π and nonbonding (n) orbitals to an antibonding orbital (denoted as σ * and π * ) with different energies. Here I will focus my attention on n→π * and π→π * transitions, since are those that I observed in measurements; I will not take into account in my manuscript the transitions that involve σ and σ * orbitals because not interesting in terms of photophysical properties and, in addition, are located usually in the far UV. Concerning this manuscript the highest observed transition is the n→π * where, upon excitation, an electron is promoted from an atom (here nitrogen) with nonbonding electrons to an antibonding π orbital. In my case the nitrogen is located in the "electron-releasing" moiety and the promoted electron goes into the lower energetic π * orbital, expected to be located in the electron-withdrawing group (using the acceptor strength as driving force). The n→π * transition is also important because it has a charge transfer character, necessary for both linear and non-linear optical applications.

One should consider that not all the transitions are allowed, indeed in absorption we have to take into account two main conditions:

 The transitions between states with different multiplicity are forbidden, while are allowed singlet→singlet and triplet→triplet transitions. Nevertheless, via spin-orbit coupling there is a weak interaction between the wavefunctions of the singlet and the triplet state, and sometimes, this transition can be observed i.e. the intersystem crossing from the first singlet-excited state to the first triplet excited state.

 Some transition can be forbidden for symmetrical reasons, for example in centrosymmetric molecules the one-photon absorption has to reach an antisymmetric state with respect to the ground state, while concerning non-centrosymmetric molecules the direct product of the irreducible representation of the ground state and of the excited state must transform as the irreducible representation of the dipole moment operator.

Taking into account that the mass of the nuclei is bigger compared with the mass of the electrons one could consider that the electronic transition occurs without changes of the nuclei position because the transition is faster compared with the nuclear motion, this is called: Frank-Condon principle, thus one can consider that the electronic transition will occur vertically. Moreover, in addition to the pure electronic transition, one can observe also the vibrational structure that depends on the vibrational levels of the excited state. Because the electronic is a vertical transition, depending on the nuclear configuration during the excitation process, the most probable transition could be between two different vibrational states, as displayed in Illustration I 2a where the most probable transition occurs between the vibrational state 0 of the ground state and the vibrational state 2 of the excited state.

Most of the time, for molecules, the vibrational structure in not observable especially when the measurements are done in particular environment such as solution or colloidal suspension. In these cases, the bandwidth depends on two effects: homogeneous and inhomogeneous broadening. The first is mostly due to the finite lifetime associated to the excited state and the existence of a continuous set of vibrational sublevels in each electronic state, while the second is due to the fluctuation of the structure of the solvation shell surrounding the chromophore. Another important feature to take into account when one speaks about absorption transition is the probability of the light absorption at a specific wavelength λ, in other words the probability that the transition occurs with a specific energy, given by the molar extinction coefficient ε(λ). This value is dependent from the electronic transition and the solvent in which is calculated from UV-visible absorption measurement using the Lambert-Beer law (Eq. I 1).

Eq I 1.

𝐴(𝜆) = 𝜀(𝜆) • 𝑙 • 𝑐

where the absorbance A(λ) measured at a specific wavelength is linearly dependent with the optical path of the light within the sample (l), with the concentration (c) and with the molar extinction coefficient (ε(λ)).

Emission of light upon one-photon excitation

Once a chromophore is excited, there are two main pathways that it can follow to return back to the ground state: radiative and non-radiative path. Between the two relaxation methods, the most interesting in terms of photophysical properties, for our purpose, is the radiative one since it involves the emission of photons. Depending on the spin multiplicity in the transition, one can call the phenomena fluorescence or phosphorescence.

As displayed in Perrin-Jablonski diagram in Illustration I 1, upon one-photon excitation the molecule can reach higher excited states (Sn) compared with the first excited state (S1); but, except few cases 4 , the emission of photons occurs from the lower excited state (called Kasha's Rule 5 ). The relaxation from Sn to S1 is a non-radiative process between electronic states with the same spin multiplicity called internal conversion. Thanks to the higher energy gap between S1 and S0 the internal conversion process is much less efficient, but indeed it can compete with the radiative decay.

Fluorescence emission band lies generally at lower energies compared with the relative absorption band, empirically observed by G.G. Stokes 6 , thanks to energy losses between the excitation and the emission combined with the Kasha's rule. There are many causes that can induces these phenomena such as fast decay to the lower vibrational level of the lower excited state, decay to higher vibrational levels of the ground state and solvent effects. Moreover, the vibrational level of the ground and the excited state are similar thus the fluorescence spectrum usually parallel the lower energetic absorption band, this is the "mirror image" rule, displayed as example in Mirror image of perylene bis-imide ligand and relative complex 7 Another possible deactivation of the excited state is displayed in Illustration I 1 as intersystem crossing (ISC) (pink wavy line, right part of the illustration). Actually, this transition is forbidden since involves the passage between two states of different multiplicity: from the first singlet excited state to the triplet state. As I wrote previously, these forbidden transitions are observable thanks to the spin-orbit coupling and the presence of heavy atoms increases the spin-orbit coupling favoring the intersystem crossing. At room temperature non-radiative relaxation from the triplet state is favored compared with the radiative T1→S0 transition called phosphorescence. This deactivation pathway is very slow, therefore the collision with solvent, or oxygen, induces preferential vibrational relaxations. However, in some cases it is possible to observe phosphorescence that lies at higher wavelength compared with fluorescence emission. This fact is explained taken into account that the lowest vibrational level of T1 is placed at lower energies compared with the first singlet excited state.

The relaxation pathway that passes through the triplet state competes with the fluorescence, decreasing the efficiency of the radiative S1→S0 transition.

The ratio between the constant relative to both radiative and non-radiative relaxation is an important data in order to study the lifetime of the excited state and consequently the fluorescence quantum yield. Indicating with k S r the radiative decay constant and with k S nr the sum all non-radiative decays constant (which include the internal conversion as well as the intersystem crossing) of the singlet state, the excited state lifetime can be obtained with:

Eq I 2.

𝜏 𝑠 = 1 𝑘 𝑟 𝑠 +𝑘 𝑛𝑟

𝑆

An important factor to take into account when one wants to compare the efficiency of different fluorophores is the fluorescence quantum yield (Φf), which is defined as the ratio of the number of emitted photons to the number of absorbed photons when the molecule is excited with a specific wavelength (Eq I 3).

Eq I 3.

𝛷 𝑓 = 𝑁 𝑒𝑚 (𝜆 𝑒𝑥𝑐 ) 𝑁 𝑎𝑏𝑠 (𝜆 𝑒𝑥𝑐 )

Eq I 4.

𝛷 𝑓 = 𝑘 𝑟 𝑠 𝑘 𝑟 𝑠 +𝑘 𝑛𝑟 𝑠 = 𝜏 • 𝑘 𝑟 𝑆 Since decay constants are complex to be measured by direct methods, the fluorescence quantum yield can be determined with various approaches including thermal and photoacoustic methods, but those considered in this manuscript are the optical methods. Φf can be measured in absolute way with an integrating sphere or relatively to a fluorescence standard 8,9 . Actually all the measurements that were done in this manuscript follow the relative determination of the fluorescence quantum yield using the Eq I 5.

Eq I 5.

𝛷 𝑓𝑆 = 𝛷 𝑓𝑅 Eq I 7.

𝑓 = 1 -10 -𝐴(𝜆 𝑒𝑥𝑐 )

Where the subscript "S" and "R" denote sample and reference respectively, Φf is the fluorescence quantum yield, n is the refractive index of the solvent, F is the spectrally integrated photon flux at the detector and f(λexc) is the absorption factor that provides the fraction of the excitation light absorbed by the chromophore.

To choose the right reference among different class of fluorophores one has to take into account that the fluorescence standard should absorb and emit within similar spectral region of the sample as well as the values of Φf of the reference and the sample should not be too different 9 . The well-established standards are three (Quinine Sulfate, Fluorescein and Rhodamine 6G) while there is a large amount of fluorophores used as references, which are less characterized or less reliable 10 , unfortunately are still missing reliable fluorescence standard within the red/near infrared spectral region, which is the region that I mainly considered in my work, as well as robust reference with low fluorescence quantum yield.

Solvent effect on fluorescence emission

In order to understand the behavior of chromophores in complex systems such as nanoparticles, it is worth studying their character by tuning polarity of the local micro/nanoenvironment that surrounds the molecule.

Depending on their nature, the interaction chromophore-solvent can strongly affect both ground and excited state, stabilizing or destabilizing them. The shift that one can observe in absorption and emission, induced by the tuning of the polarity, is called solvatochromic shift.

A compound displays a solvatochromic behavior if its absorption and/or emission bands depend on the solvent polarity. Increasing the polarity one can observe a bathochromic shift or an hypsochromic shift indicating positive or negative solvatochromism respectively.

Non-linear optical processes 11-14

Non-linear optics (NLO) is the field of optics that studies the behavior of materials interacting with very high-intensity electric field (in the order of magnitude of 10 8 V/m). Indeed, non-linear optical phenomena were observed experimentally only after the development of lasers 15 .

In linear optics the dipolar moment μ(t) is expressed in terms of permanent dipolar moment μ0, external field E(t) and the linear electric polarizability α. Eq I 8.

𝜇 ⃗(𝑡) = 𝜇 ⃗ 0 + 𝛼𝐸 ⃗⃗ (𝑡)

To generalize the dipolar moment it is necessary to expand the function with a Taylor series:

Eq I 9.

𝜇 ⃗(𝑡) = 𝜇 ⃗ 0 + 𝛼𝐸 ⃗⃗ (𝑡) + 𝛽𝐸 ⃗⃗ 𝐸 ⃗⃗ (𝑡) + 𝛾𝐸 ⃗⃗ 𝐸 ⃗⃗ 𝐸 ⃗⃗ (𝑡) +… with β first-order hyperpolarizability and γ second-order hyperpolarizability.

There are several non-linear phenomena that can be divided in second-order and third-order processes. Second-order processes, such as second-harmonic generation (SHG), sum-frequency generation (SFG) and difference-frequency generation (DFG), are observable in non-centrosymmetric systems and are directly related with β. There are two well-established ways to derive the second-order non-linear response: hyper-Rayleigh scattering (HRS) and electric-field-induced second-harmonic generation (EFISHG). I will not go in the description of these techniques since I did not use them in the work presented in this manuscript.

Among the numerous third-order non-linear processes, I would like to focus the attention on the two-photon absorption (2PA). Two-photon absorption is basically the simultaneous absorption of two photons, with same or different energy, by a single molecule. The phenomenon was theoretically predicted by Maria Göppert-Mayer during her doctoral dissertation 16,17 but the observation was possible only 30 years later with the development of lasers with the observation by Kaiser and Garret of the two-photon excitation of crystals of CaF2:Eu 2+ 18 .

Even if the topic is gaining more and more interest, reliable absolute techniques to measure the 2PA cross section (σ2) is still challenging. There are two well-established methods used for these measurements: the Z-scan 19 and the two-photon excited fluorescence (TPEF) 20 .

Z-scan method is based on the non-linear transmittance of the sample. The transmittance is measured (in so-called open-aperture condition) as a function of the intensity by scanning the sample through the focal plane of a focused laser beam. σ2 is determined with the decreasing of the transmittance in non-resonant conditions. This technique usually needs higher energy compared with TPEF that can cause the observation of other non-linear optical processes as well as strong background signal. Moreover, the Z-scan needs high concentrated solution leading to aggregation phenomena that can affect the accuracy of the measurement. The advantage of this technique is that potentially all molecules can be measured.

TPEF method is based on the measure of the fluorescence emission induced by the simultaneous absorption of two photons. The measurement is done relatively to a 2PA reference 21 , giving as a result the so-called two-photon action cross section (σ2(ω)Φf). The value that is directly obtained from the measurement is F/P 2 for each wavelength measured, where F is the fluorescence signal induced by the two-photon excitation and P is the excitation power. This indicates that fluorescence signal must have a quadratic dependence with the power, then, to produce reliable data, this dependence has to be checked for each wavelength in order to prevent excited state absorption and/or saturation of the signal, overestimating the values of the two-photon cross section. When the measurement of the sample and the reference are done as a function of the excitation power, the σ2Φf of the sample is given by the following equation (Eq I 10)

Eq I 10.

𝜎 2 𝛷 𝑓 = 𝑓 𝑅 𝑓 • 𝑛 2 𝑛 𝑅 2 • 𝑐 𝑅 𝑐 • 𝐹 𝑃 2 • ( 𝐹 𝑃 2 ) 𝑅 -1 • [ 𝑛 𝑅 𝑛 ] • (𝜎 2 𝛷 𝑓 )
𝑅 with the subscript R to indicate the reference chromophore, f is the correction factor taking into account the wavelength dispersion of the response function, n is the refractive index, c is the concentration, and the ratio in the squared brackets is the refractive index correction. From Eq I 10 it is easy to derive the two-photon cross section knowing the fluorescence quantum yield of the sample, thus to obtain reliable values of σ2, in addition to the check of quadraticity, it is necessary to have reliable value of Φf. This technique is limited to the use of fluorescent molecules but the higher sensitivity compared with Z-scan allows the measurement of low emissive chromophores unless the fluorescence quantum yield is measurable. The spatial resolution in addition with the possibility to use photons with half energy compared with the one-photon process, typically using wavelength that lies in the deep-red near infrared (NIR) spectral region, promote the two-photon absorption as suitable technique for a large variety of application in different fields such as optoelectronics 22,23 , microfabrication [24][25][26][27][28][29][START_REF] Daniel | Interfacial organization in dipolar dye-based organic nanoparticles probed by second-harmonic scattering[END_REF] , optical data storage 31,32 , photodynamic therapy [33][34][35][36][37][38][39][40][41] and fluorescence microscopy.

Bioimaging: one-photon and two-photon microscopy 42-52

Fluorescence-based imaging is a powerful tool for non-invasive study of biological processes, both in vitro and in vivo, as well as for the diagnosis of diseases.

One of the more important parameter to take into account in microscopy is the resolution, which is the minimum distance required between two objects to distinguish them. properties. This process is called photobleaching (displayed as example in Figure I 5). In confocal microscopy the excitation light is focused on the sample, therefore the probability of faster photobleaching is higher compared to the wide-field technique. Imaging in biological environment is challenging because most of the endogenous chromophores naturally present, such as NADH, tyrosine, tryptophan and FMN, absorb in the UVvisible spectral range (Table I 1). Despite the low extinction coefficient, their concentration is high enough to limit the penetration of the excitation, inducing a loss of information. In addition to this, these chromophores are also able to emit fluorescence affecting the quality of the final image. Optical windows in biological tissues [START_REF] Smith | Bioimaging: Second window for in vivo imaging[END_REF][START_REF] Hemmer | Exploiting the biological windows: current perspectives on fluorescent bioprobes emitting above 1000 nm[END_REF] To go beyond the main limits displayed by confocal microscopy, the scientific community calls in action the "world of non-linear optics". There are several techniques of microscopy that are based on non-linear processes such as SHG microscopy [START_REF] Freund | Second-harmonic microscopy of biological tissue[END_REF][START_REF] Campagnola | Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms[END_REF] , THG microscopy [START_REF] Barad | Nonlinear scanning laser microscopy by third harmonic generation[END_REF] and three-photon absorption microscopy [START_REF] Gryczynski | Three-photon induced fluorescence of 2,5diphenyloxazole with a femtosecond Ti:sapphire laser[END_REF] , but I would like to focus the attention on the technique that exploits the twophoton excited fluorescence.

Two-photon absorption process displays an intrinsic 3D resolution since the laser beam has to be focused in order to observe the non-linear process, actually the excitation volume is approximately in the order of 0. Concerning the photobleaching, one should think that in the focal point this phenomena occurs faster comparing with the confocal microscopy, since in two-photon microscopy higher photon flux are used in order to make the non-linear process allowed. Therefore, the goal is to obtain high brightness, which is defined as the product of ε or σ2 with the fluorescence quantum yield, in molecules and/or nanoobjects in order to use low power preventing the death of the cell and fast photobleaching. Since I did not use these techniques I will not go deep in the description but I guess that it is important to mention that the step further towards higher resolution is the development and the use of super-resolved fluorescence microscopies such as stimulated emission depletion (STED) microscopy [START_REF] Hell | Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy[END_REF] , scanning near-field optical (SNOM) microscopy 67 , photoactivated localized microscopy (PALM) [START_REF] Betzig | Imaging intracellular fluorescent proteins at nanometer resolution[END_REF] , stochastic optical reconstruction microscopy (STORM) [START_REF] Rust | Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM)[END_REF] 4. From bioimaging to molecular engineering 13,14,[START_REF] Lavis | Bright ideas for chemical biology[END_REF][START_REF] Albota | Design of organic molecules with large two-photon absorption cross sections[END_REF][START_REF] He | Multiphoton absorbing materials: molecular designs, characterizations, and applications[END_REF][START_REF] Escobedo | NIR dyes for bioimaging applications[END_REF][START_REF] Ptaszek | Chapter Three -Rational design of fluorophores for in vivo applications[END_REF][START_REF] Johnson | The Molecular Probes Handbook[END_REF][START_REF] Kim | Small-molecule two-photon probes for bioimaging applications[END_REF] In order to fulfill all the requirements of bioimaging in the last decades a great deal of effort was made to understand the structure-properties relationship to prepare new classes of chromophores suitable to be used as bio-probes. In this paragraph, I will try to give a short overview of the properties that the chromophores should display for quality imaging in bioenvironment.

As I mentioned before, within cells and tissues there are several endogenous absorber that could hide the desired signals. Considering two main structures of chromophores, dipolar and quadrupolar (Illustration I 4),

to improve the internal charge transfer character one can play with three particular parts of the molecule: the electron acceptor, the electron donor and the π-conjugated bridge. Specifically, enhancing the electron-withdrawing character of the acceptor group, strengthening the electronreleasing character of the donor group as well as improving the polarisability of the π bridge lead to a marked red-shift of both absorption and emission [START_REF] Genin | Fluorescence and two-photon absorption of push-pull aryl (bi) thiophenes: structure-property relationships[END_REF][START_REF] Morales | synthesis, and structural and spectroscopic studies of push-pull two-photon absorbing chromophores with acceptor groups of varying strength[END_REF][START_REF] Rouxel | Banana-shaped biphotonic quadrupolar chromophores : from fluorophores to biphotonic photosensitizers[END_REF][START_REF] Mongin | Synthesis, fluorescence, and two-photon absorption of a series of elongated rodlike and banana-shaped quadrupolar fluorophores: a comprehensive study of structure-property relationships[END_REF] . Interestingly, these three parts of the molecule play also an important role in the enhancement of the two-photon absorption process 13,14 . Indeed an The chromophores, to be used as bio-probes, have to display high one-photon and two-photon brightness in bioenvironment, thus they should show good fluorescence quantum yield associated with large ε and/or σ2 (depending on which kind of microscopy one would like to use). Unfortunately, driving the emission towards lower energies leads to a decreasing of the Φf that can be ascribed to a decreasing of the radiative decay rates in addition with an enhancement of non-radiative decay rates in the red/NIR spectral region [START_REF] Strickler | Relationship between Absorption Intensity and Fluorescence Lifetime of Molecules[END_REF] . Moreover, it is known that in biological media there is a large amount of fluorescence quenchers therefore the challenge is to prepare systems that can preserve their emissive properties in bioenvironment.

5.

Fluorescent Organic Nanoparticles: a bottom-up approach toward hyper bright nanoobjects [START_REF] Zhao | Low-dimensional nanomaterials based on small organic molecules: preparation and optoelectronic properties[END_REF][START_REF] Patra | Optical materials based on molecular nanoparticles[END_REF][START_REF] Fery-Forgues | Fluorescent organic nanocrystals and non-doped nanoparticles for biological applications[END_REF][START_REF] Wolfbeis | An overview of nanoparticles commonly used in fluorescent bioimaging[END_REF][START_REF] Battistelli | Ultra-bright and stimuli-responsive fluorescent nanoparticles for bioimaging[END_REF] "Plenty of room at the bottom" [START_REF] Feynman | Plenty of room at the bottom[END_REF] is the title of a talk presented by Richard P. Feynman in 1959 where he hypothesizes the advent of miniaturization; actually, he was the first who spoke about the "nano-world". Since then, this topic gained an overwhelming interest in the scientific community inducing the creation of a large amount of new journals that reached easily high impact factor 89 such as Nature nanotechnology (35.267), Nano letters (13.779), ACS nano (13.334) and Small (8.315).

Nano-objects are considered particles that have at least one dimension of the order of nanometer 90 . These nanoscale materials gained importance since the discovery of the so-called "nanoeffect": when the particle size is in the nanometer domain, it displays changes in physico-chemical properties of the matter such as magnetic, electric or optical properties, compared with the bulk. This Nanomaterials are suitable tools for a wide range of applications that go from optoelectronics [START_REF] Dmitri | Prospects of colloidal nanocrystals for electronic and optoelectronic applications[END_REF][START_REF] Zhai | Enhancement of 153 μm emission band in NaYF4:Er 3+ ,Yb 3+ ,Ce 3+ nanocrystals for polymer-based optical waveguide amplifiers[END_REF] to solar cells [START_REF] Robel | Quantum dot solar cells. harvesting light energy with CdSe nanocrystals molecularly linked to mesoscopic TiO2 films[END_REF][START_REF] Mora-Seró | Recombination in quantum dot sensitized solar cells[END_REF][START_REF] Lee | Highly efficient quantum-dot-sensitized solar cell based on Co-sensitization of CdS/CdSe[END_REF] , from photodynamic therapy 36,38,40,41,[START_REF] Croissant | Enhanced two-photon fluorescence imaging and therapy of cancer cells via gold@bridged silsesquioxane nanoparticles[END_REF][START_REF] Secret | Two-photon excitation of porphyrin-functionalized porous silicon nanoparticles for photodynamic therapy[END_REF][START_REF] Gangopadhyay | Fluorene-morpholine-based organic nanoparticles: lysosome-targeted pH-triggered two-photon photodynamic therapy with fluorescence switch on-off[END_REF] and drug delivery 39,[START_REF] Park | Advances in the synthesis and application of nanoparticles for drug delivery[END_REF] to bioimaging 100-105 . Among the numerous classes of nanomaterials used as bio-probes, a great effort was made in the study of semiconductor-based nanoparticles (quantum dots QDs) 101,102 , silica-based nanoparticles 40,106,107 , upconverting nanoparticles (UCNPs) 108,109 and noble metal-based nanoparticles 110 . In spite of their high photostability, tunability and brightness, these nanoparticles are known as potentially toxic nanomaterials [111][112][113][114][115] , since they are constituted by heavy metals (i.e. Cd and Te for QDs) and/or rare hearth (i.e. Yb 3+ , Eb 3+ and Tm 3+ for UCNPs). The biodegradability is still a limitation for these class of nanomaterials because they can induce serious damages to the environment. In this perspective, organic nanoparticles (ONPs) present an encouraging alternative to overcome the problems that can be encountered with "inorganic" nanomaterials, and between them, molecular-based ONPs have nowadays attracted curiosity thanks to their versatility. Potentially it is possible to prepare organic nanoparticles with any molecule one can synthesize. Therefore, one can chose carefully non-toxic organic fluorophores in order to test their ability to form nanoparticles and thus to study their behaviors. Moreover, the adaptability of organic nanoparticles allows also surface functionalization towards high selectivity targeting of bio-receptors. Contrarily to the engineering of molecules with expected photophysical behaviors, the design of ONPs is extremely challenging because one has to take into account several parameters that can strongly affect the properties of the obtained nanomaterials such as the preparation method, the size and the shape and the arrangement of chromophores upon nanoconfinement.

Preparation methods of organic nanoparticles: bottom-up and top-down approaches 117- 119

There are different methods used for the fabrication of molecular-based organic nanoparticles such as vapor deposition 120 , confined crystallization 121 , electrochemical deposition 122 but I would like to focus the attention on two other process of nanoparticles preparation: laser photofragmentation and reprecipitation.

Reprecipitation method, the easy and widely used process, can be considered as a bottom-up approach to prepare organic nanoparticles developed in the BASF laboratory 117 but firstly published by Nakanishi 123 . This process is based on fast solvent exchange producing nanoparticles under kinetic domain; basically, microliters of solution of the compound are quickly injected, usually under stirring or sonication, into a large amount of a solvent in which the compound is not soluble. There are several parameters to play with in order to control the morphology and the size of the obtained nanoobject such as the concentration of the injected solution, the use of stirring or sonication, the use of surfactants and the choice of the solvents, the good and the poor. Indeed the viscosity of the solvents and the respective diffusion have fundamental role in the reprecipitation method. Unfortunately, the nucleation/growth process is not fully understood and it is not predictable, in fact from similar compounds it is possible to obtain nanoparticles strongly different in terms of size (cf. Nevertheless, depending on the intermolecular interactions, it is possible that the molecules within the nanoparticles are organized in a short range, forming small crystalline domains.

On the other hand, laser photofragmentation (LPF) is a top-down approach to prepare nanocolloids since the idea is to form nanoparticles starting from microcrystalline powder of the compound dispersed in a poor solvent. This method was proposed by Masuhara 124-126 with a work in which is displayed the dependence of nanoparticles formation by the laser fluence used. When microcrystals are suspended in a poor solvent and exposed to intense laser pulses, this induces the breakage of the powder and the ejected particles are caught by water and stabilized as nanocolloids.

Several parameters can affect the fragmentation process leading to control both size and shape of ONPs: laser wavelength, pulse width, laser fluence, time of irradiation and total shot number.

Contrarily to the reprecipitation method, with LPF the possibility to obtain organized nanocrystals is higher since the starting powder is microcrystalline, this can be interesting in order to study the optical properties of crystalline systems in both bulk and nano domains.

Intermolecular interaction in nano-environment 127-130

The optical properties of organic nanoparticles do not show strong dependency on the size, as discussed previously for QDs and AuNPs, but they have the advantage that one nanoparticle is formed by 10 4 -10 8 molecules which induces strong additive and/or cooperative effect within the nanoenvironment. Indeed, nanoparticles display an enhancement of absorption (both one-and two-photon)

of several orders of magnitude with respect to the single chromophore.

The first studies about the behavior of molecules in confined environment were done independently in the late 1930s by Jelley 131 and by Scheibe 132 . They were the first who rationalized that molecular aggregates behave differently from the molecular solution. An extensive work was done trying to understand the effect of the intermolecular contributions on the photophysical properties, especially using distyrylbenzene derivatives 130 , and the most comprehensive theory about molecular aggregation was proposed by Kasha and coworkers 133 : the exciton model (an exhaustive description of the exciton model will be done in Chapter 3 Paragraph 1).

Upon molecular confinement especially of chromophores, which are molecules formed by more or less rigid π-conjugated systems, intermolecular π-π interactions have to be considered. Indeed, these contributions can strongly affect the emissive character of the resulting aggregates leading, most of the time, to a decrease of the luminescence properties. This phenomenon is known as "aggregationcaused quenching" (ACQ). To prevent this problem, we can call in action the molecular engineering trying to prepare chromophores with hindering groups that should increase the intermolecular distance, decreasing the probability of deleterious π-stacking interactions (several examples are discussed in the manuscript). Another way to overcome this problem was observed by Tang and coworkers 134 and the phenomenon was popularized with the name of "aggregation-induced emission" (AIE). AIE is referred to a process that exploits the decrease of the molecular motions in confined state that can lead to a decrease of non-radiative decay rate enhancing the luminescence. In fact, in the work of Tang they used methyl-pentaphenylsilole that in molecular solution display a twisted conformation while in aggregated state show a planar conformation. This phenomenon is not taking into account possible intermolecular contribution, thus is limited to the observation that a rigidification associated with a planarization of the system induce an improvement of the emissive character of the aggregates. Another phenomenon takes into account the synergic intra-and intermolecular contributions with respect to the photophysical properties: the phenomenon is known as "aggregationinduced enhanced emission" (AIEE). AIEE describes molecules that are already slightly emissive in solution but the molecular confinement induces an enhancement of the fluorescence properties thanks to the cooperative role of both intramolecular and intermolecular interactions.

Nowadays AIE or AIEE phenomena are widely exploited but, unfortunately, often wrongly claimed also due to the widespread confusion in this field.

(

Introduction

As discussed in Chapter 1, the design and synthesis of new organic chromophores with a large π-conjugated system is a very active topic thanks to the non-linear optical properties that can be exploited in a large number of different areas. In this field, great effort was dedicated to the study of dipolar "push-pull" chromophores [1][2][3][4][5][6][7][8][9] . These dyes gather in the same molecule an electron-donor and an electron-acceptor moiety, connected through a π-conjugated bridge. To tune the photophysical properties of these chromophores we can play with the degree of the charge separation in the ground state choosing appropriately the electron withdrawing and the electron-donating groups, adapting the strength of the dipole 8,[10][11][12][13] . Moreover, lengthening and/or rigidifying the π-conjugated bridge can affect the charge transfer behavior influencing consequently the photophysical properties of the chromophores 11,12,14 .

In this chapter are displayed the results of the studies that were done on push-pull dipolar chromophores bearing a triphenylamine moiety as electron-donating group (Figure II 1), which is a well known motif capable to an easy delocalization of the free doublet on the nitrogen. Moreover, triphenylamine-like compounds are known to be good 2PA systems 15 . Here will be discussed the changes of the photophysical properties that occur playing on the strength of the acceptor group;

tuning the substituent on the triphenylamine part; lengthening and/or rigidifying the π-connector bridge. Concerning the para-substituent on the triphenylamine the idea was to increase the donor character but mainly to introduce bulky groups that play an important role when the molecules are confined in nanoparticles (cf. Chapter 3). Since we wanted to play in the dipolar strength tuning the acceptor moiety is another way to improve the charge transfer. In fact, electron-withdrawing groups with increase acceptor character from aldehyde to cyanoamide, cyanoester dicyano and DETB were investigated. In the following Figure II 1 are gathered all the chromophores that will be the subject of study in this chapter. As it will be shown, the changes on the dipolar molecule will affect strongly the photophysical behavior of the chromophore as monomer, in organic solvents, as well as when confined in nanoenvironment.

Synthesis and structural characterization of dipolar chromophores

Here will be described the synthetic route that was followed to synthesize the chromophores that are the subject of study in this chapter. Since the chromophores with thienothiophene were synthesized by Cristiano Matteo Mastrodonato [CMM], I will not describe here the synthesis; I will focus the attention on the "linear thiophenes" chromophores.

Playing on the dipolar strength

To synthesize the three series of complementary chromophores, I started with the preparation of the triphenylamine precursor using a Ullmann reaction, following a procedure reported in literature 16 as shown in the Scheme II 1.

Scheme II 1.

Synthesis of triphenylamine monoiodo

A Suzuki cross coupling was used to synthesize the first aldehyde, which is the starting compound to prepare all the other chromophores of the three series. To reach the same molecule were used two different pathways displayed in the Scheme II 2 adapted from literature 17 , in both cases the compound was isolated in good yield (>75%). Because the second pathway is less expensive in terms of reagent, it was chosen to prepare the compound D1 ' a in large amount.

Scheme II 2. Synthesis of the chromophore D1 ' a through Suzuki cross coupling

Once the precursor chromophore of the three series was obtained, was prepared by a bromination using NBS (Scheme II 3a) the aldehyde of the second series; with a Suzuki coupling from this aldehyde, was obtained the compound D3 ' a as displayed in the Scheme II 3b. The procedures followed were reported in a previous work in our lab 10,11 . From both the reactions were isolated the compounds in good yields (>70%).

Through a Knoevenagel condensation using absolute ethanol or dry CH3CN (depending on the α-acid derivative) I synthesized the different members of each series starting from the respective aldehyde (1 ' a, 2 ' a or 3 ' a).The reaction condition were optimized in order to obtain the direct precipitation of the final chromophores which is not soluble in the solvent used for the synthesis, therefore the compound was isolated without further purification (for the scheme of the reaction see the Scheme II 4). To accelerate the reaction, a catalytic amount of β-alanine was used to facilitate the deprotonation of the α-position of the carbonyl. I was not able to isolate the pure chromophore D3 ' b and it was not possible to purified it easily.

Scheme II 4. Knoevenagel condensation used to synthesize the members of the three series.

Elongating the π-conjugated bridge (synthesis of the chromophore D1 ''' a)

The π-conjugated bridge of the compound D1 ''' a was synthesized starting from the commercially available fluorene in three steps. The first one is the bis-iodination of the fluorene in position 2 and 7 using strong conditions shown in Scheme II 5.

Scheme II 5. Preparation of the 2,7-diiodofluorene

To increase the solubility of the synthon and to prevent the strong intermolecular interaction that can occur in the target molecule upon aggregation, two branched iso-pentyl chain were added in the methylene bridge via alkylation as described in Scheme II 6.

Scheme II 6.

Alkylation on the methylene bridge of the fluorene

Once alkylated the bridge of the fluorene, the step further was a Kumada cross coupling to elongate the π-bridge with two thiophenes, one on each side (Scheme II 7).

Scheme II 7. Kumada cross coupling

After the Kumada coupling the π-connector of the push-pull chromophore is completed, the Like compound D1 ' c also this chromophore shows a syn-conformation of the thiophenes in the crystalline state. The torsion angle between the two thienyl groups of the bridge is 18°, while the twisting between the phenyl ring linked to the donor moiety and the thiophene is 32°.

As it can be observed in

Linear optical properties of chromophores in organic solvents

For the dipolar chromophores, subject of this chapter, the linear and non-linear optical properties in molecular solutions were studied. For an easy comprehension, because of the large amount of compounds, the chromophores are compared under three different viewpoints: the effect of the acceptor, the effect of the para-substituent and the effect of the π-conjugated bridge.

Effect of the environment

The influence of the environment on the photophysical properties was investigated by studying both absorption and emission in organic solvents, tuning the solvent polarity.

In dimethylsulfoxide. As one can see the ICT transition is not affected by the environment suggesting that the main contribution to the ground state is given by the neutral form of the molecule. It is important to notice that the chromophore with the dicyanovinyl moiety as acceptor (D3 ' d) in DMSO displays a behavior that is attributable to the retro-Knoevenagel reaction that occurs in polar solvents, which can contains traces of water that favor the hydrolysis. 20 . The positive solvatochromic behavior is coherent with an ICT transition that is associated with a large enhancement of the dipolar moment upon excitation, suggesting that the main contribution to the excited state is given by the zwitterionic form, which is stabilized by the interactions with polar solvents, therefore the excited state lies to lower energy.

The hydrolysis also strongly affects the emission. As it can be observed in Figure II 7c,e,f, the chromophores bearing strong acceptor such as cyanoester (c), dicyanovinyl (d) and DETB (e) display different properties compared to the expectations. This is an important issue that will be discussed in Chapter 3. Interestingly, the solvatochromic behavior can be fitted with a Lippert-Mataga correlation [21][22][23] as the Stokes shift are found linearly dependent on the polarity-polarisability parameter of the solvent Δf:

Eq II 1. 𝜈 𝑎𝑏𝑠 -𝜈 𝑒𝑚 = 2 ( ∆𝜇 2 ℎ𝑐𝑎 3 ⁄ ) ∆𝑓 + 𝑐𝑜𝑛𝑠𝑡 a) b) c) d) e) f)
Where νabs (νem) is the energy of the absorption (emission) maximum given in wavenumber, h is the Planck constant, c is the light velocity, a is the radius of the Onsager cavity (assumed to be spherical), Δμ is the change of the dipole moment of the solute upon excitation and

Eq II 2. ∆𝑓 = ( 𝜀 -𝑎 2𝜀 + 1 ⁄ ) -( 𝑛 2 -1 2𝑛 2 + 1 ⁄ )
where ε is the dielectric constant, n is the refractive index of the solvent.

We can estimate the value of the photo-induced change of the dipole moment of the chromophores by deriving Δμ from the Lippert-Mataga correlation (Eq II 1) using the Perrin equation 23,24 Eq II 3.

2 ( ∆𝜇 2 ℎ𝑐𝑎 3 ⁄ ) = 𝑆𝑝𝑒𝑐𝑖𝑓𝑖𝑐 𝑠𝑜𝑙𝑣𝑎𝑡𝑜𝑐ℎ𝑟𝑜𝑚𝑖𝑐 𝑠ℎ𝑖𝑓𝑡 (derived from the slope of the Lippert-Mataga correlation)

Eq II 4.

𝑎 = √𝑣 3

Eq II 5.

𝑣 = 𝑘𝑇𝜃 𝜂 ⁄

Eq II 6.

𝜃 = 𝜏 0.4 <𝑟> -1 ⁄ Table II 1.
Solvatochromism and anisotropy measurements on the chromophores of the D3' series measured in triacetin solution at 20°C. The large slope values obtained underline an expected large increase of the dipolar moment in the excited state for the chromophores. Comparing the values of Δμ in the same series (as displayed in the Table II 1), it can be noticed a marked enhancement of the photo-induced change of dipole moment passing from the aldehyde to the cyanoester (D3 ' a → D3 ' c) as acceptor group. This behavior is consistent with an increase of the dipolar strength that generates a highly polarized molecule in the excited state. On the other hand, replacing the acceptor group from c to d does not affect strongly the Δμ.

Cpd Specific Solvatochromic Shift [cm -1 ] <r> a) τ b) [ns] θ c) [ns] v d) [Å 3 ] a e) [Å] Δμ [D] D3 ' a 17

Effect induced by the tuning of the acceptor group

In this paragraph are reported the results obtained in the photophysical properties by tuning the acceptor group (from aldehyde to DETB) 25 in order to study the dependence of the properties from the dipolar strength. For an easy overview here are compared the members of three different series: D1 ' , D3 ' , D3 '' . In Table II 2 are gathered the relevant data measured for the three families of chromophores in CHCl3 solution. Chloroform was chosen as common solvent thanks to its medium-low polarity character for further comparison with the molecules confined in nano-environment.

The chromophores of these three families display an absorption band located in the visible spectral region ranging from blue to orange, depending on the acceptor. The intense band is ascribed to an intramolecular charge transfer (ICT) transition that could be associated with a combination of nπ * and π-π * transitions, while the band located in the UV spectral region can be attributed to the higher II 2), which indicates an increase of polarization of the ground state, enhancing the contribution of the zwitterionic state. One can expect that, after an increase of the electronwithdrawing strength, with a consequent enhancement of the dipolar moment, the ICT transition is more probable, leading to higher molar extinction coefficients 12 . This is not what we can observe from the Table II 2 and from the Figure II 8. While the trend of the family D3 '' follows the expectations increasing the molar extinction coefficient from 3.9 to 5.5 • 10 4 M -1 cm -1 passing from the aldehyde (CHO) to the 1,3-diethyl-thiobarbiturate (DETB), in the other two families this behavior is not observable, the εmax measured in chloroform are comparable, ranging from 3.1 and 3.7

• 10 4 M -1 cm -1
for D1 ' and between 3.3 and 3.7 • 10 4 M -1 cm -1 for D3 ' .

Table II This behavior can be ascribed to a decrease of the radiative decay rates in addition with an enhancement of non-radiative decay rates in the red/NIR spectral region 20 . It is important to underline that there is one exception among the chromophores that I presented here, in fact the chromophore D1 ' b displays an unexpected decreasing of the fluorescence quantum yield associated with a lifetime fitted with a biexponential decay. This behavior can be attributed to the presence of two different conformations of the vinyl-amide system in solution in the excited state.

Effect induced by the "para-substituent" on the triphenylamine

For an easy comparison of the effect on the photophysical properties upon the replacement of the substituent on the triphenylamine, I chose to compare two different series of compounds keeping constant the acceptor moiety: D1 ' -D3 ' c (cyanoester) and D1 '' -D3 '' d (dicyano), which could help me to give a complete overview of the behavior.

In Table II 3 In both series, the higher molar extinction coefficient is displayed by the chromophore bearing bromines as substituent, while the other two end groups lead to comparable εmax. Concerning the luminescence properties, as can be observed in Figure II 11, the behavior follows the one discussed for the absorption band. Tuning from -H to -Br is observable a blue-shift, while the substitution of -H with -tert-butylphenyl induces a bathochromic shift. However, the shift of the bands is not large indicating that the dipolar strength is only slightly affected by the replacement on the triphenylamine donor group. The bromine substituent also seem to promote efficient nonradiative decay processes (most probably intersystem crossing due to spin-orbit coupling effect) in the case of D2 '' d, which is not observable for the chromophore D2'c, leading to smaller fluorescence quantum yields and shorter lifetimes.

Effect of the tuning of the π-conjugated connector

In this paragraph is discussed the influence of the changes of the π bridge on the linear photophysical properties of the chromophores. As could be expected the nature of the connector can strongly affect the behavior of the compound. Here are compared three dipolar dyes D1 ' a, D1 '' a and D1 ''' a in order to display a complete overview of the behaviors. The important parameters to take into account when we want to compare the π bridge are the donor/acceptor character, the number of π As you can observe in Figure II 12 above, the main common constituent of the π-conjugated bridge used for the comparison is the thienyl group, which is known as good charge transmitter with a slight electron-donor character. We played on both the flexibility of the connector by replacing bisthiophenes with a thienothiophene group, and on the length of the bridge by inserting a substituted fluorene core between the bis-thiophene π systems. This is aimed to enhance the linear and non-linear response of the chromophore.

In the Table II 4 are gathered the relevant photophysical data measured in organic solution using chloroform as common solvent.

Non-linear optical properties of chromophores in organic solvents

Thanks to the emissive properties of these dipolar chromophores, we were able to estimate the two-photon cross section (σ2) by measuring the two-photon brightness (σ2Φf) with the two-photon excited fluorescence (TPEF) technique widely discussed in the introductive chapter. The measurements were done in a range between 680 nm and 1160 nm using as references Fluorescein (from 680 nm to 1000 nm) or Nile Red (from 1000 nm to 1160 nm). The range of detection was chosen depending on the λmax 1PA as well as the range of emission.

For an easy comprehension, this section will be divided in the same paragraphs that were used in the previous section: effect of the acceptor part, effect of the "para-substituent" and effect of the πconnector. To simplify and make a complete overview, here will be displayed the non-linear optical properties of the chromophores used for the linear characterization. Since the chromophores are solvatochromic, to have a good comparison with the results obtained upon one-photon excitation, the 2PA measurements were done in chloroform.

Effect induced by the tuning of the acceptor group

As is displayed in Figure II 14 (one member for each series was taken as an example) and in Table II 5, the dipolar chromophores show an intense and structured two-photon absorption band in which the maximum wavelength is almost twice the λmax 1PA suggesting that the ICT transition is symmetrically allowed upon both one-photon and two-photon excitation. This behavior is expected for non-centrosymmetric "push-pull" chromophores.

Effect induced by the "para-substituent" on the triphenylamine

As observed in the previous paragraph, the chromophores display an intense two-photon absorption band related to the same transition observed in the linear process (Table II 6). The trend observed parallel the one already discussed in the previous paragraph, an increase of the dipolar strength induces an enhancement of the σ2 associated with a bathochromic shift. As observed for the linear process, the shift caused by the replacing of the para-substituent on the donor moiety is not marked, as you can observe in

Effect of the tuning of the π-conjugated connector

In the Table II 7 which is almost twice the λmax 1PA . This was not expected for a dipolar "push-pull" chromophore, because it is a typical character for centrosymmetric dyes. Actually, this behavior can be due to another electronic transition that is more allowed upon one-photon absorption than upon two-photon absorption, thanks to the presence of the fluorene core. On the other hand, D1 ' a and D1 '' a display the expected behavior.

It is worthy to underline the fact that comparing D1 ' a and D1 ''' a, each chromophore displays a broad structured 2PA band in which the sub-bands are superimposable with different relative intensity.

Moreover, the addition of the fluorene induces a strong enhancement of the two-photon cross section, paralleling the behavior observed upon one-photon excitation. This hyperchromic effect is associated to a marked blue shift due to the higher energetic most allowed two-photon transition. The rigidification of the π-conjugated bridge, passing from thienothiophene to bis-thiophene, induces a hypsochromic shift. Contrarily to what was observed for the linear process, this effect is now associated with a hypochromic effect.

Conclusion

In this chapter were discussed the changes of both linear and non-linear optical properties in a series of complementary "push-pull" chromophores, by tuning the chemical structure playing on the electron-withdrawing or electron-releasing strength of the end groups, as well as the properties of the π-conjugated bridge.

All dyes display an intense one-photon absorption band associated with a strong ICT transition with high molar extinction coefficient, up to 5.5 • 10 4 M -1 cm -1 . Moreover, the studied molecules show a tunable emission band ranging from green (i.e. D1 '' a) to red (i.e. D1 ' c and D1 '' d) or even NIR (i.e. The changes of the structure allowed us also to tune the two-photon absorption band in the range between 700 nm to 1150 nm enhancing the two-photon cross section up to 2000 GM in molecular solution.

In The "nano-world" is gaining an overwhelming role thanks to the versatility of the nanoobjects, especially when we speak about organic nanoparticles with which we can synthesize molecules with expected properties in order to obtain nanoaggregates with specific behaviors. In this chapter will be described the morphological and both linear and non-linear optical properties of fluorescent organic nanoparticles, so called FONs, prepared starting from dipolar chromophores discussed in Chapter 2.

The main peculiarity of dipolar molecules is their tendency to form dipole-dipole interaction in aggregated state, which can include highly concentrated solution, nanoparticles and solid state. The normal arrangement for dipoles is the so-called antiparallel configuration as displayed in Illustration III 1; this distribution of chromophores leads to strong intermolecular interaction. This antiparallel arrangement of the molecules decreases strongly the overall polarization of the system, moreover it can also cause an enhancement of intermolecular charge transfer transition. Antiparallel configuration usually affects strongly the photophysical properties of the molecular aggregates, therefore including nanoparticles. This behavior is well explained by the theory of molecular exciton 1,2 .

Once you approach two molecules they start to interact, therefore you have a system with four combined states in which two of them are degenerate.

Scheme III 1. Schematic representation of the excitonic splitting in a dimer where M1 and M2 are the two dipoles that form the dimer, * represent the excited molecules and 2J is the energetic difference between the two splitted states

The combinations between the two degenerate states provoke the excitonic splitting, depending on the arrangement of the molecules; there is a state at higher or lower energy compared with the starting state. The transition moment is given by the vector that is the sum of each transition dipole moment of the dimer, therefore when we have the limit "parallel" form, so-called H-aggregates (Scheme III 2a), the allowed state will be the one at higher energy, thus we should observe a hypsochromic shift of the ICT band associated with no luminescent properties. When we have the limit "in-line" form, so- As it can be observed in Scheme III 3, depending on the angle between the two dipole moments is allowed either the lower |e ' › or the higher |e '' › state, influencing the photophysical properties. Experimentally, it was observed that both states are reached, indicating a more complex aggregate in which the molecules are arranged with different relative angles between them.

The dipolar chromophores studied in this chapter are molecules based on triphenylamine as electron releasing group. This moiety was chosen, in addition to what was discusses in the previous chapter, also because of the typical propeller shape that could prevent close π-stacking interactions between neighbor chromophores. Moreover, the replacing of the para-substituent on the donor group, in addition to the changing of the donating strength already discussed, allow us to study the behavior of the chromophores with bulky groups within the nanoparticle, preventing the interchromophoric interaction, which are deleterious for emissive properties, and increasing the colloidal and chemical stability [5][6][7] .

The starting idea was to prepare full organic, biocompatible fluorescent nanoparticles (FONs)

with an emission range that lies in the biological transparency window (cf. Chapter 1), with high onephoton and two-photon brightness in order to use them as fluorescent probes in bioimaging.

Preparation of nanoparticles with their structural and morphological characterization

To prepare organic nanoparticles (ONPs) was used the easy and fast method of nanoprecipitation, a process based on solvent exchange. A stock solution of chromophore, with mM concentration, was prepared in a solvent fully miscible with water (such as THF, Acetone or DMSO) and a small amount of the stock solution was injected in a large amount of water (typically 1% of stock solution), in which the compound is not soluble. This process induces a fast nanoaggregation of molecules depending mainly on the nucleation/aggregation process but also on the diffusion of the solvent in water 8,9 .

Scheme III 4.

Schematic representation of the nanoprecipitation process

After the preparation, the FONs were characterized by transmission electron microscopy (TEM) to study the size distribution and the shape, as well as by zetametry to study the surface potential, important parameter to predict the colloidal stability overtime (that will be discussed later in the chapter). In Table III The evolution of nucleation/growth is of main importance in the nanoaggregation process, and is not predictable. As displayed in As one can see from TEM images in Figure III 2, the shape of the obtained nanoparticles is spherical; therefore, we could assume that the nanoobjects prepared are not crystalline. This was expected since the solvent exchange is a kinetic process and the chromophores do not have enough time to the full thermodynamic organization within the object. Anyway, the ONPs are not completely amorphous, indeed a previous work of the lab 10 has shown that on the surface of the nanoparticle, which is in contact with water, the chromophores are organized in a short range while going deeper in the bulk the molecules increase their disorder.

Linear and non-linear optical properties of FONs in water

After the confirmation of the nanoparticles formation, it was possible to go further and characterize the obtained nanoobjects under the viewpoint of optical properties. Thanks to the luminescent properties of the prepared ONPs, it was also possible to estimate the two-photon cross section of the chromophores upon nano-confinement.

Due to the large number of samples, for an easy and clear comparison the FONs will be compared in the same way used as for the chromophores dissolved in organic solvents.

To In Table III 2 are gathered the relevant data measured for the members of the three families of chromophores upon nanoaggregation.

The FONs of these three series display a broad and intense absorption band that lies from the violet to the green visible spectral region depending on the acceptor group. As already discussed in the previous chapter, the intense band located at lower energies is ascribed to an ICT transition, while the high energetic band is attributed to π-π * transition. The ICT band is associated with a large molar extinction coefficient ranging from 2.3 to 5.0 • 10 4 M -1 cm -1 , but a more exhaustive discussion, which include also the comparison with the results obtained for the chromophores in solution, will be found Indeed, tuning the acceptor group it was possible to obtain nanoobjects that cover the spectral range from yellow to NIR. Concerning the fluorescence lifetime, one can see in Table III 2, that all the FONs display two lifetimes; the shorter lifetime, ranging from 0.9 (D3 '' a) to 1.6 nm (D1 ' b) is dominant with respect to the other one, which is longer (from 3.0 to 4.9 nm). In each sample, the short lifetime can be attributed to the "shell", the surface, which is in contact with water and where it is possible to have competitive non-radiative decays; on the other hand, the long lifetime can be attributed to the bulk of the particle.

Two-photon characterization

The Concerning the luminescent properties, it is worth to highlight the behavior of these FONs: as expected, replacing the hydrogen with bromine induces a hypsochromic shift of the emission band but, strikingly, no bathochromic shift was observed by adding the bulky tert-butylphenyl group. Indeed, as reported in Table III 3 Also in this case, as observed in the previous paragraph, the fluorescence decay can be fitted with a biexponential decay giving two fluorescence lifetimes. The shorter and dominant lifetime is attributable to the chromophores that form the surface, while the longer one can be ascribed to the bulk of the nanoparticle.

Two-photon characterization

As could be expected, the properties of the chromophores upon nano-confinement in water are comparable with the behaviors observed for the chromophores in solution. As displayed in Figure III

Effect of the tuning of the π-conjugated connector

One-photon characterization

In this paragraph FONs prepared with chromophores D1 ' a, D1 '' a and D1 ''' a are compared.

Since the connectors between the end-groups are not similar here I cannot generalize the properties, I will just give an overview of the different behaviors that can be induced by the change of the π-bridge.

As reported in Table III 4

Two-photon characterization

Similar to what was observed for the chromophores in solution, when the molecules are confined in nano-environment it can be noticed that in FONs prepared with D1 ' a and D1 '' a the same transition is allowed in both one-photon and two-photon process, as displayed in Figure III 12a-b.

However, contrarily to solutions, FONs prepared with D1 '' a show higher two photon cross section, suggesting an arrangement of chromophores within the nanoparticles that improve the non-linear response. As one can observe in Figure III 13, the trend followed by the different molecules in solid state as well as in nanoparticles is not the same for all the samples. Indeed, it can be noticed that tuning the chemical structure we have the emission of FONs similar (as in the case of D3 ' c), or redshifted (as in the case of D1 ''' a, D1 ' c and D2 ' c) compared with the solid state, suggesting the different arrangement of the molecules in both states.

Behavior of the chromophores in solution and nano-environment

It is interesting to compare the behavior of the chromophores in both solution and nanoparticle in order to study the influence of aggregation on the photophysical properties. Since these chromophores are solvatochromic, I will not discuss precisely the shift of both absorption and emission band. I can say that for all chromophores we observed an hypsochromic shift of the Moreover, it can be observed either a broadening or an asymmetric shape of the absorption band when the molecules are confined in FONs compared with the molecule in solution. This effect can be ascribed to excitonic coupling, which is favored by the interchromophoric interactions between neighbor molecules within the nanoparticle. As you can notice, the hyperchromic or hypochromic effect upon nanoaggregation depends on the structure of the chromophore, therefore is not possible to generalize the behavior.

Non-linear optical properties

As can be expected, the molecular confinement of two-photon responsive chromophores strongly affects their 2PA behavior. Indeed, dipolar interactions and consequent arrangement of molecules when confined in nano-environment influence the two-photon response of polar and polarizable chromophores 16,17 . As expected, the confinement of dyes within a nanoparticle induces a marked decrease of the two-photon cross section per chromophore subunit, as discussed for the linear As one can notice, the trend followed upon two-photon excitation does not parallel the one observed in one-photon absorption. This underlines the fact that predicting the behavior of chromophores in nanoparticles is not easy.

One-photon and two-photon brightness

As widely discussed in the introductive chapter, to have hyper-bright nanoobject is a great issue, especially if we would like to use our systems as bio-probe in confocal and/or multiphoton microscopy. The brightness of nanoparticle takes into account the number of chromophoric subunits that form the object, therefore the molar extinction coefficient and the two-photon cross section for FONs were estimated taken into account the concentration of nanoparticles, and not the moles of chromophore in nanoparticles.

In spite of the low fluorescence quantum yield of the nanoparticles due to the aggregation caused quenching process, one can observe in Table III 5, that our FONs display huge one-photon (up to 2.5 • 10 8 M -1 cm -1 ) and two-photon brightness (ranging from 0.02 to 1.4

• 10 6 GM) surpassing all a) b) c) d)
other types of nanoparticles including QDs [18][19][20] , depending also on the cooperative/additive effect due to the number of chromophores per nanoparticle (See Table III 1). Since the brightness is strictly dependent of the size of the nanoparticle, and so far it is not possible to predict the size and the shape of the FONs, the brightness of the nanoobjects is not predictable either. Our systems are strongly bright with a tunable emission ranging from green to NIR, therefore they could be nice tools for further applications in both one-photon or two-photon microscopy.

Colloidal and chemical stability of FONs overtime

Another important behavior that we should consider when we speak about FONs is the stability of the colloidal suspension overtime. This property can affect strongly the possibility of nanoparticles application in bio-environment.

This behavior is connected with the value of the surface potential, the more the nanoparticle displays a negative surface potential, the less is the probability of nanoparticles aggregation overtime.

As reported in Table III 1 To study the colloidal stability, the evolution of both absorption and emission spectra was monitored overtime.

As one can observe in Figure III 16 that passing from D ' a → c, so increasing the electron withdrawing character of the acceptor group, there is an enhancement of the colloidal stability. In fact D1 ' a display a marked decrease of the absorption band and a quenching of the luminescent properties after one week, while D1 ' c after one month only shows a slight decrease of both absorption and emission spectra indicating the improvement of the stability. Parallel behavior can be observed adding the tert-butylphenyl groups in the available para position of the triphenylamine (D1 ' a → D3 ' a), these bulky groups improve considerably the overtime stability as expected 6 .

This observation suggests that the stability is dependent from the chemical structure of the chromophores. Strong electron withdrawing as well as bulky substituent lead to an improvement of the stability. In Chapter II Paragraph 3.1 (Effect of the environment) it was mentioned that chromophores with strong acceptor group could undergo hydrolysis. Since the condensation reaction is an equilibrium, if the product molecule is surrounded by water, the reaction could return back towards the reagents. Actually, a retro-Knoevenagel reaction was observed for some chromophores in polar solvents, such as acetone, acetonitrile or DMSO (cf. Figure II 6-7). After these observations, one could think that a chromophore that could undergo a retro-condensation in a water environment like that of FONs, should not be stable at all, therefore we should not be able to obtain nanoparticles of these chromophores. Strikingly, a very interesting behavior was observed, as displayed in Figure III 18. We were able to obtain nanoparticles and, indeed, FONs were very stable in comparison with the chromophore in DMSO after addition of a drop of water. The stability of these nanoaggregates suggests us a cooperative shielding effect that protect the chromophores within the nanoparticle from the "aggressive" water environment that could induce the retro-Knoevenagel reaction.

Conclusion

In this chapter were displayed the preparation and the morphological characterization of fluorescent organic nanoparticles prepared starting from dipolar chromophores. We were able to obtain colloidally stable spherical nanoparticles with an average size ranging from 21 nm to 66 nm.

On these FONs were studied the optical properties upon one-photon and two-photon excitation. By tuning the chemical structure, it was possible to tune the photophysical properties, as already observed for the chromophores in organic solution. We were able to obtain nanoparticles with a tunable emission from green to deep red or even near infrared, associated with an aggregationcaused quenching phenomenon, indeed the fluorescence quantum yield of FONs is low compared with that measured in CHCl3 solution. In spite of the low Φf (up to 0.14 for green-yellow emitter D2 ' a), all

FONs display a huge one-photon (ranging from 0.02 • 10 8 to 2.5 • 10 8 M -1 cm -1 ) and two-photon (ranging from 0.02 • 10 6 to 1.1 • 10 6 GM) brightness, which associated with the two-photon excitation and the emission range in the biological transparency window, suggesting these nanoparticles suitable to be used for both confocal and multiphoton microscopy.

Moreover, an interesting cooperative shielding effect was observed in FONs that prevent the hydrolysis for chromophores bearing strong acceptor groups.

CHAPTER 4-CHARGED DIPOLES 1. Introduction

In the last decade, a large effort was made to understand the cellular internalization of nanoobjects [1][2][3][4][5][6][7][8][9][10] , while the knowledge is still limited concerning the distribution of nanoparticles within the bio-environment in relation with their morphological and structural properties. Indeed, the interaction of nanoparticles with cells in vitro and in vivo is known to be strongly affected by their size and their shape [11][12][13][14][15][16][17][18][19] and numerous studies shown that the surface potential plays a significant role in cellular internalization [20][21][22][23][24] . For instance, positively charged nanoparticles are usually internalized faster than the neutral and negative one. Moreover, it is interesting to explore this way because the membrane of cancer cells are mostly negatively charged thanks to anionic biomolecules in the double layer.

So far, the surface potential of FONs prepared in the hosting lab was always negative [25][26][27][28][29][START_REF] Daniel | Interfacial organization in dipolar dye-based organic nanoparticles probed by second-harmonic scattering[END_REF][31][32] .

Therefore, our challenge was to try to prepare organic nanoparticles with a positive surface potential in order to compare the behaviors in bio-environment. In literature, there are few examples of organic nanoparticles prepared with organic salts 33,34 , between them the most known class of nanomaterials are the so-called nanoGUMBOS (Group of Uniform Materials Based on Organic Salts) proposed by the group of Warner [35][36][37][38][39][40] .

To prepare positively charged nanoparticles our idea was to use methyl-pyridinium and methyl-quinolinium as acceptor group in dipolar chromophores with a similar structure compare to the dyes displayed in Chapter 2 and 3, with a triphenylamine as donor moiety and thienyl group in the πconjugated path, using a lipophilic and hindering counterion (Scheme IV 1). Heteroaromatic salt-type acceptor moieties display large electron-withdrawing character that leads to interesting optical properties. Moreover, methyl-pyridinium and methyl-quinolinium salts are gaining more and more 106 interest in recent years thanks to their potential application in medical fields [41][42][43] and in optoelectronics materials 44 .

Scheme IV 1. Dipolar pyridine, quinoline, methyl-pyridinium and methyl-quinolinium derivatives subjects of this chapter with the resonance structure of the hemicyanine species (DIp + specifically)

Optical properties of chromophores in organic solvents

The photophysical properties of p + and q + chromophores in solution are compared with the respective neutral precursors in order to have terms of comparison.

Effect of the environment

Solvent polarity can strongly affect the photophysical response of chromophores and that is why also for this new series of compound the solvatochromic behavior was studied. Toluene, THF, DCM and acetone were chosen as common solvents in order to dissolve the dyes as well as preventing protonation of the pyridine and quinoline moiety, which influences the optical properties.

It is interesting to notice the different behavior of the chromophores of the two classes of the series: the neutral dipoles and the hemicyanine. In ICT band, this behavior is known in literature 45,46 . Concerning the emission one can observe that a tuning of the solvent polarity does not affect strongly the fluorescence, non-solvatochromic emission is typical for this class of compounds 46,47 . can be explained by the separation of the ion pair in polar solvent, actually this behavior can be seen also in THF for the compound DIIp + .

Photophysical characterization in THF solution

As common solvent, was chosen THF for the full photophysical characterization of these chromophores. The relevant data are gathered in Table IV 1.

Table IV All chromophores display a broad absorption band that lies between the violet and the greenyellow visible range depending on the dipolar strength of the dyes. The band can be ascribed to a strong ICT band associated with a large molar extinction coefficient ranging from 2.7 to 4.7 • 10 4 M -1 cm -1 . An elongation of the π-conjugated path by adding one thiophene in the bridge (I → II) induces a bathochromic shift (Figure IV 2a) of the ICT band associated with the expected hyperchromic effect 32,48 , while the enhancement of the electron withdrawing character by changing the acceptor group from p to p + leads to a marked shift towards longer wavelengths with a decrease of the molar extinction coefficient, contrarily to what was expected 27,48 (see also Chapter 2). Anyway, increasing both the electron withdrawing character of the acceptor and the elongation of the π-conjugated path induces a broadening of the absorption band. It is interesting to underline the different behavior displayed by chromophores after the replacement of p with q and p + with q + (Figure IV 2b-c). The elongation of the conjugation leads to a shift of the ICT band towards lower energies, it can be observed that this behavior is more pronounced in hemicyanine chromophores thanks to the presence of a formal charge on the acceptor group.

Moreover, it can be observed a decreasing of the εmax after substitution p → q and p + → q + . The luminescent properties generally parallel the behavior observed in absorption, the elongation of the bridge by adding a second thienyl group induces a bathochromic shift; passing from DIp to DIIp we have a decrease of the fluorescence quantum yield, while going from DIq to DIIq induces a slight enhancement of Φf. Enhancing the strength of the acceptor group after alkylation of the nitrogen in the heteroaromatic rings (p → p + and q → q + ) leads to a marked red-shift associated with a broadening of the band. Moreover, it is interesting to highlight that the hemicyanine chromophores synthesized are near infrared emitters; unfortunately, these chromophores display also a marked quenching of the luminescent properties (Table IV 1) indicating that the relaxation mainly follows non-radiative pathways. As it can be seen in Figure IV 2b-c, the extra ring in the electron acceptor moiety (p → q and p + → q + ) induces a shift towards longer wavelengths as observed for the absorption band.

Figure IV 3. Comparison between emission spectra of different chromophores studied in this chapter

Concerning the fluorescence lifetime, it is worth to underline that the neutral dipolar chromophores display a mono-exponential decay with a lifetime between 0.73 and 1.98 ns, while charged compounds show bi-exponential fluorescence decay.

Photophysical characterization of hemicyanine dyes in SDS micellar solution

Since the idea is to use these chromophores in bio-environment, one of the fundamental requirements is water solubility. Unfortunately, these dyes are not soluble in water, thus I tried to use a The inner pocket of the micelles formed with SDS has a hydrophobic character therefore the chromophores dissolved in SDS display an hypsochromic shift compared with THF solution, even though the environment is water (Figure IV 4a-b). This blue shift is more pronounced for the emission band, which is associated with a large enhancement of the luminescent properties, for example one can see in Table IV 2 that the Φf of compound DIIp + , from THF to SDS, displays an increase of more than 20 times (from 0.005 to 0.11) in the NIR region.

Strikingly, contrarily to what was observed in THF solution, an elongation of the π bridge does not affect the ICT transition band, which is strongly affected by the change of the acceptor group from the methyl-pyridinium to the methyl-quinolinium. On the other hand, both structural changes induce a shift of the emission band towards lower energies, as observed for the chromophores dissolved in THF solution.

Optical properties of chromophores when confined in nano-environment

The step further was the preparation of nanoparticles with these classes of chromophores to study the behavior of the molecules upon nanoaggregation. The preparation method used was the same as used and described before: the nanoprecipitation.

Morphological and structural characterization of FONs

After their preparation, the nanoobjects obtained were characterized by transmission electron microscopy, for the size and the shape, and by zetametry to have an idea of the surface potential of these nanoparticles. Unfortunately, as one can see in Table IV 3

Photophysical characterization of hemicyanine dyes upon nanoconfinement

The FONs prepared with methyl-pyridinium and methyl-quinolinium as acceptor group display an intense and broad absorption band that lies in the blue-green visible spectral region. The molar extinction coefficient (gathered with the relevant photophysical data in Table IV 4) of the chromophores after nanoaggregation is comparable to, or even higher than the one measured in THF solution. This behavior can be due to a cooperative effect induced by the packing of the molecules in confined environment.

It is worth to notice the absorption band of FONs DIIp + , whose unusual shape may be due to the arrangement of chromophores and counter ions within the nanoparticle. As observed for the chromophores dissolved in THF or SDS, the ICT band is not affected by the change of the electron-withdrawing moiety, while it can be observed a bathochromic shift of the emission band passing from DIIp + to DIIq + . The shift towards low energies is observed also with elongating the π-conjugated pathway.

Despite their low luminescent properties, it is worth to underline the fact that these FONs display strikingly high one-photon brightness (up to 2.2 • 10 7 M -1 cm -1 ), thanks to the high molar extinction coefficient estimated per nanoparticle, that associated with the NIR emission make these object suitable for confocal microscopy.

Conclusion

In this chapter, the photophysical properties of hemicyanine chromophore in organic solution and in SDS micellar solution were discussed. It was shown that the methylation of the nitrogen of the heteroaromatic rings induces a large bathochromic shift of the emission band yielding deep red to NIR emissive chromophores. This behavior is associated with a marked quenching of the fluorescence quantum yield.

We were able to prepare spherical organic nanoparticles, ranging between 46 and 82 nm size, with a positive surface potential. Despite their low Φf and their poor stability overtime, they show an interesting large one-photon brightness, up to 2.2 • 10 7 M -1 cm -1 , in the NIR spectral region; the step further will be to study these nanoobjects in bioenvironment as fluorescent probes. Moreover, the two butyl chains form a cross-like structure with the core (89° the angle between the main plane of the core and the plane of the alkyl chain), which is consistent with literature 6 .

The hindering groups prevent short π-π interactions, in fact among neighbor molecules one

Effect of the environment

The influence of the environment on the photophysical properties was investigated by measuring both absorption and emission in organic solvent increasing the solvent polarity, from cyclohexane to dimethylsulfoxide.

As one can observe in Figure V 5, the absorption spectra measured for the chromophores are not affected by the solvent. Since the dipole is short and the dipolar strength is not large, this behavior was expected, suggesting that the main contribution to the ground state is given by the neutral form. On the other hand, an increase of the solvent polarity strongly affects the emission band inducing a marked bathochromic shift associated with a broadening of the band, from cyclohexane to DMSO (See Figure V 6). Interestingly, as it can be predicted that chromophores 1Mono and 1Tris are solvatochromic, for both dyes 1Bis the behavior is not expected. However, the latter compounds show the typical solvatochromic behavior of standard "push-pull" chromophores undergoing an increase of dipole moment upon excitation. This is similar to what was observed for some quadrupolar chromophores that display symmetry breaking in the excited state [7][8][9] . This attitude can be ascribed to the presence of two "independent" dipolar subunit simply connected by a π-conjugated bridge that works only as a rigid connector.

Full characterization in THF solution

To study the photophysical properties of the articulated dipoles THF was used as common solvent, for a later comparison with the molecules upon nanoaggregation.

As one can observe in Table V 1 Tuning the acceptor group one can predict a bathochromic shift going toward stronger dipoles (1Bis → 2Bis). The observed red-shift is associated with a marked hyperchromic effect (εmax up to 7.8

• 10 4 M -1 cm -1 ) as could be expected by increasing the dipolar strength and enhancing the probability

c) d)
of the ICT transition. It is worthy to underline that a similar behavior is observed in chromophores 2Bis, in which a rigidification of the π system (BP to F) induces an hyperchromic effect and a bathochromic shift. shows a red-shift. The emission bands are located in the green spectral region with medium-low fluorescence quantum yields for green emitters ranging from 0.11 (1Bis(F)) to 0.39 (1Tris). A decreasing of the luminescent properties can be observed in the quadrupolar-like chromophores 1Bis, which can be consistent with the rotation motion of the system as non-radiative deactivation pathway.

In fact, as it can be observed in Table V 1, the non-radiative decay rate of chromophores 1Bis is much higher than that of 1Mono and 1Tris. Similar to what was observed for absorption, the emission band is strongly affected by the tuning of the acceptor group. Indeed, replacing the aldehyde with a dicyanovinyl group induces a marked shift towards lower energies attributable to the increase of the dipolar strength. This bathochromic shift is associated with a quenching of the luminescent properties, indeed the chromophores 2Bis(F-BP) display a Φf lower than 0.01. This marked decrease of the emission could be explained by the molecular motion that affects more the chromophores with dicyanovinyl group as acceptor moiety with respect to the CHO. More interesting are the anisotropy results for chromophore 1Bis(F) and 1Tris, which can be qualitatively explained by the excitonic model. Due to the small interaction between the dipoles, the splitting between the one-exciton states is small. Nevertheless, when exciting in the blue edge of the absorption band, we mainly populate the higher-energy one-exciton state, while upon excitation in the red edge of the absorption band we mainly populate the lower-energy one-exciton state. Since emission always arises from the lowest-energy excited state, fluorescence anisotropy amounts to 0.4 upon excitation in the red edge, while it depends on the relative polarizations of the two one-exciton states when exciting in the blue edge. A similar interpretation can be given for the trimer, the decrease of anisotropy when moving the excitation wavelength from the red to the blue edge of the absorption band is faster because in this case we have three one-exciton states with different polarizations.

Non-linear optical properties

We were able to estimate the two-photon cross section of these chromophores by measuring the two-photon brightness with the 2PEF technique. Since this method is based on the emission, it was not possible to measure reliable data of chromophores 2Bis(F) and 2Bis(BP) in THF because of their low fluorescence quantum yield. Like for the molar extinction coefficient, also the two-photon cross section is affected by the number of dipolar subunits within the same molecule. In fact, as can be observed in Figure V 10a, the increase of the number of dipoles from 1Mono to 1Tris induces a hyperchromic effect, up to 225 GM.

Anyway, contrarily to the one-photon process, the enhancement is not linear. Strikingly, chromophore 1Bis(BP) displays a higher σ2 max than the respective dye with fluorene as connector.

. FONs preparation and morphological characterization

To prepare organic nanoparticles with the articulated dipoles the nanoprecipitation method described in Chapter 3 was used.

After preparation, the size and the shape of FONs were characterized by transmission electron microscopy (TEM) and confirmed by atomic force microscopy (AFM), while the surface potential was measured by zetametry. The relevant data concerning the characterization of nanoaggregates are reported in Table V 3 As discussed in Chapter 3, it is not possible to predict the size and the shape of FONs depending on the chemical structure of the chromophore. Indeed if one take as example nanoobjects prepared with dyes 1Bis(F) and 1Bis(BP), one can observe that the size of FONs prepared with 1Bis(BP) is almost twice larger and there is almost one order of magnitude of difference between the number of molecules that form the nanoaggregates (1.1 • 10 4 and 9.6 • 10 4 respectively). Moreover, the NPs presented herein display spherically shaped FONs with size ranging from 30 nm up to 70 nm. Table V 4). This shift towards low energies is also observable for the emission band that, from the blue-green region, shifts to the red visible region. Concerning the fluorescence quantum yields, one can observe from the comparison between the chromophore in THF solution and in FONs, that the aldehydes display comparable or slightly lower Φf, while a very small enhancement of the quantum yield is noticed upon aggregation for dyes 2Bis(F) and 2Bis(BP). This phenomenon indicates that the deleterious π-π intermolecular interaction are prevented by the hindering groups present in the chemical structure of the chromophores, as well as the rotational motions are decreased by aggregation, which increase the radiative decay enhancing the emissive properties of the dyes in FONs. This interesting behavior was not observed for simple dipolar "push-pull" chromophores presented in Chapter 3 and Chapter 4, thus it is a proof that the molecular arrangement of multi-dipolar chromophores upon nanoaggregation leads to a decrease of the non-radiative processes that compete with luminescence properties.

All nanoparticles display two fluorescence lifetimes: the longer one can be attributed to the fluorescence decay of the bulk, while the shorter one is ascribed to the lifetime of the chromophores that form the surface, in contact with the water environment, which leads to competitive non-radiative decays.

From the direct comparison between the chromophore in THF solution and upon nanoaggregation, one can observe in Figure V 15 an interesting behavior: even if upon nanoconfinement there is a decrease of εmax (except for FONs prepared with dye 2Bis(BP)), one can observe a broadening of the ICT band associated with a slight hyperchromic effect towards longer wavelengths. This behavior can be ascribed to an excitonic coupling that occurs in confined environment. Both the luminescent properties and the excitonic coupling observed for these chromophores upon aggregation suggest that the arrangement of dyes within the nanoparticle is not purely of Jaggregate type 10,11 .

Two-photon characterization

Thanks to the enhancement of the luminescent properties of chromophores 2Bis within FONs it was possible to estimate the two-photon cross section by measuring the TPEF on these samples and reliable data was obtained. The behavior of FONs is similar to that of the dyes in THF solution: λmax 2PA ≈ 2λmax 1PA thus the ICT transition is allowed upon both one-photon and two-photon excitation (see

Figure V 16a-b).
Since observed this behavior was also observed for nanoparticles prepared with 1Bis and 2Bis chromophores, it is interesting to underline that even in confined environment the communication between dipoles through fluorene or biphenyl is negligible.

The comparison of the two-photon responses of FONs prepared with 1Mono, 1Bis(F) and 1Tris reveals a different behavior with respect to what was observed in solution. Moreover, the trend is also different if considering the effect upon one-photon excitation: the two-photon cross section measured upon nano-aggregation seems to be not affected by the number of dipoles within the same 147 molecule. Indeed, as displayed in Figure V 16c, FONs prepared with 1Bis(F) show higher two-photon response than 1Tris. We can attribute this behavior to the arrangement of chromophores within nanoparticles that produces a less polarizable environment, which is more sensitive to the two-photon excitation. Nevertheless, when the chromophores are confined in nanoparticles, the highest σ2 max is also displayed by chromophore 1Bis(BP). section, except for FONs prepared with 1Bis(F) which exhibit higher σ2 max compared with the chromophore dissolved in solution.

In spite of the decrease of the two-photon response, it is worthy to underline that the 2PA spectra of FONs display a shoulder towards low energies, suggesting an excitonic coupling. According to these observations, we can hypothesize the presence of an intermediate arrangement of chromophores within the nanoparticle that allows both transition bands.

Colloidal and structural stability overtime

To study the colloidal stability, as previously described, the evolution of both absorption and emission spectra of FONs was evaluated overtime. Thanks to their negative surface potential, ranging from -54 to -68 mV, these nanoparticles are expected to show a high colloidal stability. As one can

Bioimaging and cytotoxicity of FONs

Multicolor imaging is a powerful technique in order to study complex phenomena such as protein localization 12 , live cell internalization of proteins 13 or simultaneous observation of different lymphatic channels 14 . This method is based on the introduction in the bio-environment of two or more fluorescence probes and it is necessary that these markers display good overlap between the excitation spectra while they have to show different emission range in order to distinguish the different functions that are studied. Organic fluorophores are intensively used in multicolor imaging thanks to the wide range of possible structures that can be prepared and their biocompatibility. On the other hand, their photostability and brightness in biological media are very low.

The confinement of the articulated dipoles within nano-environment induces a cooperative/additive effect that can strongly affect the brightness of FONs. Indeed, in spite of their f) e) low fluorescence quantum yield, these nanoobjects shows giant one-photon (up to 14.8 • 10 8 M -1 cm -1 ) and two-photon (ranging from 0.2 to 5.0 • 10 6 GM) brightness, as displayed in Table V 6, suggesting these nanoparticles as suitable tools for confocal and multiphoton imaging. Therefore, their potential application was investigated in vitro. In order to exploit the possibility to do multicolor imaging we chose 1Bis(BP) as green emitting FONs and 2Bis(BP) as red emitting FONs, and COS 7 cells as a model system. Indeed their small size could lead to an uptake of the nanoparticles involving both clathrin-mediated endocytosis 15 as well as passive internalization, also due to their soft character 16,17 .

The two FONs have spectral properties favorable for simultaneous multicolor fluorescence imaging: good overlap between the absorption bands, large stock shift and well separated emission bands. brighter than the red one. Thus, we provided tools for multicolor imaging in biological environments by using probes of similar characteristic (size, surface charge, composition). The targeting of biological species of interest with these nanoparticles will allow simultaneous visualization, localization and study of their interaction. It was previously shown in the hosting laboratory the potential of similar FONs for multicolor single-particles tracking in live cells 18 .

In view of the potential application of the described FONs for bioimaging, we further The following equations (Eq. V 1-2) describe the electronic Hamiltonian and its two eigenstates:

Eq V 1. 𝐻 = ( 0 -√2 𝑡 -√2 𝑡 2 𝑧 0 ) Eq V 2. { |𝐺〉 = √1 -𝜌 |𝑁〉 + √𝜌 |𝑍〉 |𝐸〉 = √𝜌 |𝑁〉 -√1 -𝜌 |𝑍〉
The parameter ρ measures the weight of the zwitterionic state in the ground state, and thus defines its polarity. Dipolar chromophores are considered neutral in ground state when 𝜌 < 0.5 and zwitterionic when 𝜌 > 0.5. In the limit case in which 𝜌 = 0.5, the system displays two basis states (neutral and zwitterionic) that coexist with the same weight. This case is the so-called: "cyanine limit" 23,24 .

Thanks to the interesting properties observed experimentally for chromophores 1Mono, 1Bis(F) and 1Tris, the idea was to describe firstly the behavior of the chromophore 1Mono as a The program that I used during a period that I spent in Parma University was written by Prof.

Francesca Terenziani (Università degli Studi di Parma). This program takes into account different parameters that can be divided in three parts:

 Electronic part  Vibrational part  Interaction with solvent
A more detailed description of the parameters used can be found in Table V To describe the system, two different approaches were used for the fitting. In the first strategy, electron-phonon coupling was considered and eight phonon states were used for each dipolar unit to reach the convergence in the calculated spectra. In the second approach, purely electronic states were used, neglecting the effects of molecular vibrations: this allowed simplifying and accelerating the calculations. In particular, for trimer 1Tris calculations accounting for electron-phonon coupling are too computationally costly and slow to be processed. Nonetheless, it was possible to fully describe the behavior of 1Mono and 1Bis(F) with the vibronic part.

Description considering the electron-phonon coupling

The essential state model is a semi-empirical method, therefore I had to fix the parameters to reproduce the previously displayed experimental data, specifically absorption and emission spectra in solvents of different polarity and the 2PA spectrum in THF. The parameters fixed correspond to a highly neutral dye, with 𝜌 ≅ 0.05, consistently with non-solvatochromic absorption and strongly solvatochromic emission.

Once the parameters were set for the "monomer", the idea was to keep all of the terms fixed for the "dimer" 1Bis(F), while adding the distance and mutual orientation of the two dipolar units.

In order to describe the intermolecular interactions in the "dimer", the following parameters are required: the length of the dipole (5.6 Å according to the crystal structure), the distance between the dipoles (9.7 Å according to the crystal structure) and the angle between the two dipoles.

Concerning the angle, it was used the crystallographic torsion angle between the two dipoles: 172.5°.

However, this value did not allow to obtain the correct excitation anisotropy spectrum, which could instead be well reproduced with an angle of about 110°. While the specific value of the angle has huge effects on the anisotropy spectra, allowing to have a reliable estimation of the mean mutual orientation, absorption and fluorescence spectra are slightly influenced by the specific choice of this angle. Indeed, it was verified that the spectra obtained for the angle extracted from the crystal structure, 172.5°, have negligible differences with respect to those obtained for 110°.

As written just before, we wanted to keep the values fixed for chromophore 1Mono to reproduce the experimental data of 1Bis(F) (see Table V 8). Actually we were forced to change the value of z0 passing from 1Mono to 1Bis(F), as to reproduce the slight red-shift experimentally observed in the ICT band. In fact, independently of the distance and mutual orientation of the two dipolar subunits, the specific structure of the dimer does not allow to obtain attractive interactions between the two dipoles, so that only blue-shifts can be expected as a result of inter-chromophore interactions. The decrease of z0 is consistent with slightly modified dipolar units, in particular a longer conjugation length. In Figures V 22 is displayed the comparison of experimental and calculated spectra with respect to the solvatochromic behavior. The agreement is good, especially concerning the position and shape including inhomogeneous broadening; as one can observe the vibronic structure in cyclohexane is also well reproduced. We focused our attention on the n-π * transition band, therefore the shoulder that can be ascribed to a π-π * transition was not taken into account in the calculation.

Furthermore, there is a good quantitative agreement between the experimental and the calculated 1PA spectra in THF, thanks to the fact that the molar extinction coefficient follows a linear hyperchromic dependency (see Figure V 23). A marked deviation is observed for the 2PA spectrum of the dimer that experimentally is less than twice the one of the monomer. This behavior is not justified in absence of strong interactions.

Description without considering the electron-phonon coupling

In this case too, it was necessary to optimize of the parameters for chromophore 1Mono since we want to use this dye as prototype molecule.

For chromophore 1Bis(F) interaction parameters were the same as previously discussed (dipole length, distance and angle), while for 1Tris the parameters were extracted from the crystal structure: the length of the dipole is 5.6 Å, and the distance between the dipoles is 12.2 Å.

As can be observed in It is important to notice that the angle of 110° corresponds to the mean mutual orientation of the dipolar units, while a distribution of angles is possible in the sample; indeed, even if the medium is vitrified, a static disorder is present. Moreover, the absence of source of inhomogeneous broadening in our calculations forced us to increase the homogeneous linewidth of the vibronic transitions in order to obtain broad fluorescence excitation spectra at low temperature (T = 90 K, which is estimated to be the glass transition temperature of 2-MeTHF 25 ).

Conclusions

In this chapter were discussed the linear and non-linear optical properties of a new class of multichromophoric molecules, bearing from one to three dipoles connected by rigid/semi-rigid πconjugated bridge, in both molecular solution and nanoparticles. The changes of the photophysical properties of these chromophores were studied by enhancing the acceptor strength by tuning the electron-withdrawing end-group from CHO to DCV.

The optical characterization suggests the low/non-communication between the different subunits within the same molecule, and this hypothesis was confirmed by the essential state model calculation done in collaboration with Prof. Francesca Terenziani from Parma University.

We were able to obtain spherical nanoparticles with an average diameter ranging from 30 to 70 nm with excellent colloidal and chemical stability. Interestingly, the molecules upon aggregation display similar or even higher (2Bis(F-BP)) Φf compared with the chromophores in THF solution;

moreover the prepared FONs displayed a broadening of the two-photon absorption band ascribed to a pronounced excitonic coupling that occurs in confined environment.

Despite their low fluorescence quantum yield, the FONs display large one-photon and twophoton brightness with low cytotoxic activity, evaluated through MTT test after 24h of incubation in COS7 cells. other hand, in non-centrosymmetric molecules the transition between the ground and the final state is allowed both upon one-photon and two-photon excitation; moreover quadrupolar chromophores, compared with the dipolar analogue, display higher two-photon cross section thanks to their peripheryto-core charge redistribution upon excitation [1][2][3] . In the last decades quadrupolar-like molecules were intensively studied [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21] , the goal is to design and synthesize quadrupolar systems with strong emission and large two-photon brightness (σ2Φf) in the red-NIR region. As discussed earlier for dipoles, also for quadrupoles the shift of both absorption (one-photon and two-photon) and emission towards lower energies depends on the endgroups and the core as well as the conjugation of the connecting bridge. Considering the same donoracceptor couple (at least with similar strength), a longer π-conjugated path is needed to obtain strong quadrupolar red-NIR emitter, compared with dipoles. In the confined state, this may lead to a higher probability of interchromophoric interaction causing a decrease of the emissive properties.

Illustration
This chapter reports a systematic study on quadrupolar chromophores, displayed in Figure VI

1, using triphenylamine as common donor end-group and a benzothiadiazole (BTDA) as electronwithdrawing core by either tuning the length and the conjugation of the bridge or playing with the strength of the core adding a second BTDA moiety.

Linear photophysical characterization in organic solution

For an easy comprehension, the chromophores of this chapter are compared under two different points of view: the change of the photophysical properties induces by both the π-conjugated path and a modification of the core by adding a second BTDA.

Effect of the π-conjugated bridge

In this paragraph are discussed the results obtained measuring the photophysical properties on quadrupoles by changing the π connector between the two donor end-groups and the acceptor core, in order to compare how the bridge can affect the optical behavior. We played with both the rigidity of the π system as well as the extension of the conjugation by adding an ethynyl, a vinyl or a thienyl linker to a fluorene. In Table VI 1 are gathered the relevant data.

The absorption spectra can be clearly divided in two main bands, the more intense band is located at lower wavelength and can be ascribed to a high energy π-π * transition, while the less intense band is red shifted and belongs to an internal charge transfer transition, the considered molar extinction coefficient is related to the ICT band. It is displayed in The luminescence properties do not follow the trend observed for the absorption. Indeed It can be observed a shortening of the fluorescence lifetime with passing from the chromophore with f as π-bridge to the elongated ones. Moreover, one can notice that the substitution of the double bond with a thienyl moiety does not affect the lifetime of the excited state, while replacing the vinyl with the ethynyl linker leads to a marked decrease of the τ1.

Effect of the core

Another way to increase the charge transfer character of molecules is to try to enhance the strength of the donor or the acceptor moiety 1,2 , to do so, we tried to increase the periphery to core charge transfer by adding a second electro-withdrawing group in the core of the quadrupole.

As observed in the previous paragraph, the most intense band is related to a highly energetic π-π * transition, while the more interesting internal charge transfer band is lying at lower energies.

Indeed, both chromophores display a broad ICT absorption band located in the blue spectral region.

They do not show significant changes related to the addition of the second BTDA such as a shift of the ICT band or changes of the molar extinction coefficient. Probably the strengthening of the core does not affect the ground state leading in no strong differences, except a slight hypochromic effect with passing from B1 to B2. On the other hand, the addition of a second benzothiadiazole induces a slight bathochromic shift of the emission band (QfeB2 λmax em = 718 nm) associated with a marked decrease of the luminescence quantum yield (see Table VI 2). The marked shortening of the lifetime of the excited state (2.20 ns → 0.79 ns) suggests that the relaxation pathway mainly follows non-radiative decays.

Non-linear photophysical characterization in organic solution

Thanks to their emissive properties, we were able to evaluate the two-photon cross section of these quadrupolar chromophores by measuring the two-photon excited fluorescence. Due to their emission, from orange to NIR, the measurements were done in a range between 750 nm and 1160 using Fluorescein and Nile Red as reference chromophores 28 .

Effect of the bridge

The chromophores studied in this paragraph display intense two-photon absorption spectra with the two-photon cross section ranging from 880 GM up to 2972 GM. As discussed in the introduction of the chapter, the 2PA band is markedly blue-shifted in relation with the linear ICT band, underlying that upon two-photon excitation the reached excited state is different compared with the one-photon transition.

Effect of the core

These chromophores display a structured intense two-photon absorption band with the maximum markedly blue-shifted compared with the double of the λmax 1PA as expected (cf. Paragraph 1: Introduction). Interestingly, in both 2PA spectra is visible a shoulder located at low energies that can be ascribed to the one-photon allowed transition. The addition of the second BTDA in the core of the molecules does not affect the absorption band in terms of shift, but it is interesting to underline the unexpected decrease of the σ2 at λmax 2PA measured passing from B1 to B2, while is observable a slight hyperchromic effect at low energies. The latter behavior is probably related to the one-photon allowed transition more pronounced in the chromophore with double core.

FONs preparation and morphological characterization

The nanoparticles were prepared with the already described nanoprecipitation method. The size and the shape of the obtained nanoaggregates were evaluated using the transmission electron microscopy (TEM), while the surface potentials were measured by zetametry. In The prepared FONs display a spherical shape with a diameter size ranging from 19 nm to 57 nm. Since the nanoparticles are spherical, one can assume an internal disorder, and so no specific interactions should occur between neighbor chromophores. Furthermore, as widely discussed earlier in the manuscript, the number of chromophores per nanoparticles is an important data in order to evaluate the overall brightness of the nanoobject and this number is directly related to both the size of the nanoparticles and the molar weight of the molecule.

Linear and non-linear optical characterization of FONs

The next step, after the preparation of FONs was to study their photophysical properties in order to see if these nanoobjects can be used as probes in bioimaging. Also in this paragraph the description will be divided taking into account the conjugated path and the strength of the core.

Effect of the π-conjugated bridge

One-photon characterization

Following the trend observed for these chromophores in solution, the elongation of the conjugation also induces a bathochromic shift of the absorption band upon nanoaggregation.

Concerning the molar extinction coefficient, we have a parallel behavior: a marked hyperchromic effect from QfB1 to QfeB1 and then to QfvB1, while we observed a decrease of the εmax by replacing the vinyl linker with a thienyl moiety.

Two-photon characterization

Within nanoparticles, the molecules have a similar behavior than that already observed in solution: as expected, when the molecules are confined in nano-environment, the excited state reached with the non-linear process is different compared with the one reached upon one-photon excitation.

Contrarily to what was observed in one-photon absorption, the comparison between the spectra measured for the quadrupoles in THF solution and in FONs in water did not show the cooperative effect that enhance the two-photon cross section at the maximum wavelength, except for nanoparticles prepared with QfB1. However, all the dye within nanoparticles display a broadening of the absorption band with the formation of a shoulder (marked for QfeB1 and QftB1), towards low energies, suggesting the excitonic coupling also observed in the linear process. Moreover, the comparison also shows that the sub-bands of the 2PA spectra are well superimposed between solution and nanoparticles, but with relative intensity changes: typically the most intense band for the nanoconfined quadrupole is red-shifted with respect to the chromophore in THF.

Two-photon characterization

The molecules upon nanoconfinement and the one dissolved in organic solvent display similar properties. Indeed, as shown in Figure VI 13a, the maximum wavelength of the two-photon absorption spectra is markedly blue-shifted compared with twice λmax 1PA , indicating that, even in nanoparticles, the excited state reached upon two-photon excitation is not the one reached after the absorption of one photon.

Biological studies

As widely discussed in the previous chapters, the confinement of a large number of chromophores in nanoparticles induces additive/cooperative effects that lead to high one-photon and two-photon brightness. We were able to test the nanoparticles prepared with QfvB1 and QfeB2 in COS7 cells in order to study their ability of internalization and their luminescent properties in bioenvironment. The images were taken after 24h of incubation of the FONs in cells using DAPI as label of nuclei.

Conclusions

In this chapter were discussed the optical properties of new red-emitting quadrupolar dyes in both molecular solution and nanocolloidal suspension. The tuning of the photophysical properties was obtained by playing on the length and rigidity of the π connector between the two donor end-groups and the benzothiadiazole acceptor core, as well as by increasing the electron-withdrawing character of the core adding a second BTDA moiety.

The comparison between the photophysical properties measured in solution and nanoparticles suggest that upon nanoconfinement the arrangement of the molecules induces a general hyperchromic effect of both molar extinction coefficient and two-photon cross section, indicating an excitonic coupling behavior. I demonstrate that a careful molecular design leads to chromophores with expected properties, preventing strong molecular interaction, deleterious for the emissive properties, and improving the colloidal and structural stability of the nanoobjects prepared. Moreover, was displayed an additional/cooperative effect of the chromophores upon nano-confinement, leading to large molar extinction coefficients as well as large two-photon cross section of FONs.

Comparing these nanoparticles with the commercially available chromophores 1 or nanoobjects 2,3 it can be noticed that the prepared FONs display at least one order of magnitude higher brightness (one-photon brightness: QDs ≈ 10 5 M -1 cm -1 ; Carbon Nanodots ≈ 10 3 M -1 cm -1 ;

Nanodiamonds ≈ 10 7 M -1 cm -1 ; Alexa 647 ≈ 10 5 M -1 cm -1 ; two-photon brightness: QDs ≈ 10 4 GM).

The final purpose is to use these systems in biological environment for either one-photon or two-photon microscopy. Since some of the FONs display large one-and two-photon brightness associated with an emission in the red/NIR region, which falls in the biological transparency window, it is possible to use these nanoparticles in bioimaging using COS7 cells as bio-environment. The nice results obtained open new perspective for future works such as the functionalization of these nanoobjects in order to target specifically some organelles of the cells for theranostic purposes. Monocrystals suitable for X-ray diffraction were obtained by slow evaporation of a THF solution.

ABBREVIATIONS

Melting Point: degradation over 178°C. Following the general procedure using D2 ' a as aldehyde and methylcyanoacetate as α-acid derivatives to yield compound D2 ' c as bright red solid (yield = 81%).

Melting Point: 226°C. Melting Point: 224°C. was added n-Bu4NBr (3.75 mmol, 0.2eq.) and the mixture was stirred at RT for 15 minute. When the time was over, at the reaction mixture were added 2 (19.3 mmol, 1 eq.), 1-bromo-3-methylbutane (115 mmol, 6 eq.) and 20 mL of toluene. The mixture was stirred at 65°C for 2h, then it was cooled down to RT and left reacts for 24h more. The reaction was quenched with water and extracted with dichloromethane. The organic phase was dried under reduced pressure. The crude was purified by a column chromatography (SiO2, petroleum ether). A dark olive solid was isolated (yield = 46%). 1.2 eq.), K2CO3 (0.383 mmol; 2.5 eq.) and Pd(dppf)Cl2 (7.65•10 -3 mmol; 0.05 eq.) were placed in a Schlenk flask with a stirring bar. After drying with a mechanical pump, 10 mL of 1/1 mixture toluene/Methanol degased with Ar were added in the flask. The temperature was increased to 85°C and kept for 24h. After slow cooling to RT, the reaction mixture was filtered over celite and washed with dichloromethane. The filtrate was evaporate under reduced pressure. The crude was purified with chromatography column (SiO2, dichloromethane/petroleum ether, 70/30), the dark yellow powder was recrystallized from acetone. A bright yellow microcrystalline powder was isolated (yield = 60%).

Melting Point = 155°C

Monocrystals suitable for X-ray diffraction were obtained by slow diffusion of pentane layered over a solution of CH2Cl2.

Melting Point = 161-162 °C.

1 . 14 4 20 5. 1 .2 2 . 3 . 2 . 2 .properties of chromophores in organic solvents 2 . 1 . 1 .

 1142012322211 Absorption of ultraviolet and visible light 1.2.Emission of light upon one-photon excitation 1.3.Solvent effect on fluorescence emission 2 Non-linear optical processes 10 3 Bioimaging: one-photon and two-photon microscopy From bioimaging to molecular engineering 18 5 Fluorescent Organic Nanoparticles: a bottom-up approach toward hyper bright nanoobjects Preparation methods of organic nanoparticles: bottom-up and top-Synthesis and structural characterization of dipolar chromophores 40 2.1.Playing on the dipolar strength 2.2.Elongating the π-conjugated bridge (synthesis of chromophore D1 ''' a) Effect induced by the tuning of the acceptor group 3.3.Effect induced by the "para-substituent" on the triphenylamine 3.4.Effect of the tuning of the π-conjugated connector 4 Non-linear optical properties of chromophores in organic solutions 59 4.1.Effect induced by the tuning of the acceptor group 4.2.Effect induced by the "para-substituent" on the triphenylamine 4.3.Effect of the tuning of the πstructural and morphological characterization of nanoparticles 3 Linear and NLO properties of FONs in water 3.1.Effect induced by the tuning of the acceptor Effect induced by the "para-substituent" on the triphenylamine 3.2.1. One-photon characterization 3.2.2. Two-photon characterization 3.3.Effect of the tuning of the π-conjugated connector Behaviour of the chromophore in solution and nanoenvironment 4.2.Non-linear optical properties 4.3.One-photon and two-photon brightness 5 Colloidal and chemical stability of FONs overtime Effect of the environment 2.2.Photophysical characterization in THF solution 2.3.Photophysical characterization of hemicyanine dyes in SDS micellar solution 3 Optical properties of chromophores when confined in nano-environment 3.1.Morphological and structural characterization of FONs 3.2.Photophysical characterization of hemicyanine dyes upon nanoconfinement 3.3.Monitoring overtime of colloidal and structural stability of FONs preparation and morphological characterization 4.2.One-photon characterization 4.3.Two-photon characterization 4.4.Colloidal and structural stability overtime 5 Bioimaging and cytotoxicity of FONs 6 Articulated dipoles: an essential state model description 6.1.Description considering the electron-phonon coupling 6.2.Description without considering the electron-phonon coupling 6.3.Anisotropy in vitrified Me-

Illustration I 2 .

 2 Potential energy diagram with vertical transition (Franck-Condon principle)

Figure I 1 .

 1

Figure I 1 .

 1 Figure I 1.Mirror image of perylene bis-imide ligand and relative complex7

Figure I 2 .

 2 Figure I 2. Solvatochromic behavior of a) neutral dipole, b) charged dipole and c) quadrupole

Illustration I 3 .

 3 Two-photon excited fluorescence (right panel) compared with the one-photon excited fluorescence (left panel) Since the process is not probable, the laser beam has to be focalized in order to enhance the two-photon absorption transition; therefore, the non-linear process occurs in the focal point. Moreover, in 2PA processes there is a quadratic proportion between the energy that is absorbed from the molecule and the intensity of the incident light, these two characters leads to improve the 3D spatial resolution as displayed in Figure I 3.

Figure I 3 .

 3 Figure I 3. Difference between the spatial resolution in one-photon (upper part, λexc = 405 nm) and two-photon (lower part, λexc = 1050 nm) process

  Usually in fluorescence microscopy the x-y resolution is approximately half of the excitation wavelength ranging from 200 to 400 nm for visible light, while the depth field, which is the thickness along the optical axis layer, depends on the microscopy technique that we consider. In wide-field fluorescence microscopy, the sample is all excited and the depth field is approximately 2-3 μm. In fact the detector collects also the out-of-focus fluorescence, thus generating blurred images (Figure I 4). To overcome this problem was developed another technique: the confocal microscopy. The main differences between wide-field and confocal microscopy are the light source (which is a laser) and the use of a pinhole that prevents the out-of-focus fluorescence detection. Contrarily to the wide-field, where the entire image is collected, in confocal microscopy the sample is scanned, therefore the first technique allows live images and videos while with the second does not. One important improvement of the confocal microscopy is the possibility to observe different slices of the sample with a resolution of about 500 nm, allowing the achievement of 3D images.

Figure I 4 .

 4 Figure I 4. Thick section of fluorescently stained human medulla observed in a) wide-field fluorescence and with b) confocal microscope (revised from ref.53)

Figure I 5 .

 5 Figure I 5. Photobleaching of fluorescein and Texas Red dye in cells. Cytoskeleton of cells stained with fluorescein and Texas Red dye, excited with 488 or 594 nm lasers for A)t = 0 s B)t = 5 s and C)t = 15 s 54 .

Figure I 6 .

 6 Figure I 6.Optical windows in biological tissues[START_REF] Smith | Bioimaging: Second window for in vivo imaging[END_REF][START_REF] Hemmer | Exploiting the biological windows: current perspectives on fluorescent bioprobes emitting above 1000 nm[END_REF]

  photon absorbers as one can see in Figure I 7 (σ2 ranging from 0.04 GM of NAD(P)H to 0.45 GM of LipDH)[START_REF] Blab | Two-photon excitation action cross-sections of the autofluorescent proteins[END_REF][START_REF] Huang | Two-Photon Fluorescence Spectroscopy and Microscopy of NAD(P)H and Flavoprotein[END_REF] , thus this technique allows high sensitivity thanks to very low background signal and

Figure I 7 .

 7 Figure I 7. Two-photon action cross sections and emission spectra from a basis set of biological molecules. (a) two-photon brightness of six molecules that contribute much of the intracellular 2PE intrinsic fluorescence. (b) Emission spectra of the compounds shown in (a) 65

  only citing a few. Interestingly, this topic was awarded with the Nobel Prize in chemistry in 2014 in the person of Eric Betzig, Stefan W. Hell and William E. Moerner. The super-resolution techniques are aimed to break the diffraction resolution limit in order to obtain better images (Figure I 8).

Figure I 8 .

 8 Figure I 8. STED microscopy (circular inset image) compared to a conventional light microscope (outer image) image of filament structures within a nerve cell. (G. Donnert, S. W. Hell; Max Planck Institute for Biophysical Chemistry 70 )

  To overcome this problem, the fluorophores used as probes in bioimaging should absorb and emit in the biological transparency window for preventing autofluorescence, allowing the deeper penetration as well as decreasing the cell damage. In the last decades a large work was done in order to understand the relationship between the molecular design and the bathochromic shift of the optical properties: generally an enhancement of the ICT character of the chromophore should induces a bathochromic shift of optical properties. Illustration I 4. Representation of a) general dipolar chromophore and b) general quadrupolar chromophore. Indicating with A and D the electron acceptor and the electron donor moiety respectively.

  ICT character of the considered chromophore generally leads to an enhancement of the two-photon cross section.For the preparation of new dyes for bioimaging, another difficulty that can be encountered is the possibility of re-absorption. This process occurs when the Stokes shift is short and the chromophore displays a large overlap between the absorption and the emission band. As one can observe in Figure I 9, that shows the overlap between absorption and emission spectra of two commercially available bioprobes (Alexa Fluor ® 488 and Alexa Fluor ® 700), the chromophores display a short Stokes shift associated with a large overlap between the absorption and the emission band.

Figure I 9 .

 9 Figure I 9. Absorption (dashed line), emission (solid filled line) and excitation/emission filters (colored bars) of Alexa Fluor ® 488 (green) and Alexa Fluor ® 700 (red) 81

  behavior can be easily observed in semiconductor-based nanoparticles (Figure I 10a) or in noble metal-based nanoparticles (Figure I 10b) where slight changes of the particle size and/or shape induces significant changes of photophysical properties.

Figure I 10

 10 Figure I 10. a) Cartoon, photograph, and emission spectra showing the color changes of quantum dots (QDs) with increasing nanocrystal size 116 , b) Gold nanoparticles (AuNPs) commonly applied in biomedical applications: gold nanorods, silica-gold core-shell nanoparticles and gold nanocages. The color of these nanoparticles varies with aspect ratio, shell thickness and/or galvanic displacement by gold 110 .

Chapter 3 Paragraph 2 ,

 32 Chapter 4 Paragraph 3 and Chapter 5 Paragraph 4). Since the nanoparticles are prepared under kinetic domain the probability to produce amorphous nanoobject is quite high.

Figure II 1 .

 1 Figure II 1. Dipolar push-pull chromophores (n = 1, 2 or 3)

  Scheme II 3. a) Bromination of the para position of the diphenylamine (compound D2'a); b) Suzuki cross coupling to achieve the chromophore D3 ' a.

Figure II 2 Figure

 2 Scheme II 8.Mono-formylation via Vilsmeier-Haack reaction

FigureFigure

  Figure II 4. a) D2 ' b in general position with the solvent of crystallization in the crystal lattice b) Dimerization of the chromophores through weak hydrogen bond c) Interaction between two parallel layers

Figure II 5

 5 Figure II 5. a) D1 ''' a in a general position b) Packing between two layers c) Pile of chromophores

Figure II 6

 6 is displayed the comparison between the absorption spectra measured for two chromophores of the series D3 ' (a and d) increasing the solvent polarity from cyclohexane to

Figure II 6 .

 6 Figure II 6. Comparison between the absorption spectra measured for the chromophores a) D3 ' a and b) D3 ' d, increasing the solvent polarity

Figure II 7 .

 7 Figure II 7. Comparison between the emission spectra measured increasing the solvent polarity for the chromophores a) D3 ' a; b) D3 ' c; c) D3 ' d; d) D3 '' a, e) D3 '' d and f) D3 '' e.

  energetic π-π * transition. As one can notice, in Figure II 8, this transition is emphasized in the families D3 ' and D3 '' thanks to the presence of the tertbutyl-phenyl as para-substituent in the triphenylamine moiety. From the Figure II 8 one can clearly observe that an enhancement of the dipolar strength induces a marked bathochromic shift of the ICT band, associated with a narrowing of the absorption band (Table

Figure II 8 .

 8 Figure II 8. Comparison between the one-photon absorption spectra measured for the different members of the dye family a) D1 ' ; b) D3 ' and c) D3 '' in CHCl3 solution.

Figure II 9 .

 9 Figure II 9. Comparison between normalized absorption (solid line) and emission (dashed line) spectra measured for the different members of the dye family a) D1 ' ; b) D3 ' and c) D3 '' in CHCl3 solution (consistent color code between the figures of the paragraph)

  are gathered the relevant data measured for the two series of chromophores in CHCl3 solution. Both families display an intense ICT band placed in the cyano-green spectral region and as already discussed beforehand, this transition is associated with a large molar extinction coefficient up to 5.4 • 10 4 M -1 cm -1 , as it can be observed in Figure II 10 and in Table II 3. Basically the idea is similar to the one explained previously, an enhancement of the push-pull effect by increasing the strength of the dipole, induces a bathochromic shift of the absorption transition. Indeed, as one can observe in Figure II 10, the addition of two bromine atoms in paraposition on the triphenylamine donor moiety induces an hypsochromic shift related to the slight electron-withdrawing inductive effect. On the other hand, the replacement of the hydrogen with a tertbutylphenyl group leads to a bathochromic shift of the ICT transition thanks to the slight electronreleasing behavior. As one could expect, an enhancement of the dipolar strength should induce an hyperchromic effect of the absorption band although is not what was observed (see Figure II 10).

Figure II 10 .

 10 Figure II 10. Comparison between the one-photon absorption spectra measured for the different members of the series a) D1 ' -D3 ' c; b) D1 '' -D3 '' d in CHCl3 solution.

Figure II 11 .

 11 Figure II 11. Comparison between the normalized absorption (solid line) and emission (dashed line) spectra measured for chromophores a) D1 ' -D3 ' c; b) D1 '' -D3 '' d in CHCl3 solution (consistent color code between the figures of the paragraph)

  Figure II 12.

Figure II 12 .

 12 Figure II 12. Dipolar push-pull chromophores changing the π-conjugated bridge between the endgroups.

  As displayed inFigure II 13a the rigidification of the connector induces a hypsochromic shift associated with a slight hyperchromic effect due to a higher transmission character. The shift towards lower wavelength can be due to the lower delocalization of the charge in the thienothiophene π-D1'''a D1'a D1''a conjugated bridge compared with the bis-thiophene. On the other hand the addition of the fluorene core within the two thiophenes provokes a marked enhancement of the molar extinction coefficient (from 3.1 • 10 4 D1 ' a to 5.1 • 10 4 D1 ''' a) associated with a blue-shift of the ICT band. Concerning the fluorescence properties, interestingly, by tuning the bridge towards higher rigidity, one can observed a marked blue-shift of the emission band associated with a slight increasing of the fluorescence quantum yield. As displayed in Table II 4 this structural change does not affect strongly the fluorescence lifetime as well as the radiative decay rate. While, the elongation of the conjugation by adding the fluorene induces a broadening of the emission band. The elongation induces also a marked decrease of the fluorescence quantum yield associated with an enhancement of the nonradiative decay rate compared with the chromophore D1 ' a.

Figure II 13

 13 Figure II 13. a) Comparison between the one-photon absorption spectra measured for the chromophores D1 ' -D1 ''' a in CHCl3 solution b) Normalized absorption and emission spectra measured for the chromophores D1 ' -D1 ''' a in CHCl3 solution.

Figure II 14 .

 14 Figure II 14. Superimposition between one-photon (dashed line) and two-photon (colored point and solid line) absorption spectra of chromophores a) D1 ' c; b) D3 ' d and c) D3 '' e in CHCl3 solution.

Figure II 15 .

 15 Figure II 15. Comparison between the two-photon absorption spectra measured for the different members of the family a) D1 ' ; b) D3 ' and c) D3 '' in CHCl3 solution.

Figure II 16 .

 16 Figure II 16b, the higher and more shifted band belongs to the chromophore bearing the tert-

Figure II 16 .

 16 Figure II 16. Comparison between the two-photon absorption spectra measured for the different chromophores bearing the same acceptor moiety a) c (cyanoester); b) d (dicyanovinyl) in CHCl3 solution.

  are gathered the relevant data measured for the series of chromophores D1a in CHCl3 solution. Interestingly, as you can observe in Figure II 17c, chromophore D1 ''' a display a structured absorption in which the most allowed transition band is markedly blue-shifted compared with twice the wavelength of the ICT band upon one-photon process. A band can be also observed at 840 nm, a) b)

Figure II 17 .

 17 Figure II 17. Superimposition between one-photon (dashed line) and two-photon (colored point and solid line) absorption spectra of chromophores a) D1 ' a; b) D1 ''' a; c) D1 ''' a in CHCl3 solution and d) comparison between the 2PA spectra measured for the different members of the series D1a.

D3 ' d

 d and D3 '' e) associated with a high fluorescence quantum yield, even for the NIR dyes (i.e Φf = 0.55 D1 ' d and Φf = 0.24 D3 ' d).

Figure II 18

 18 is displayed a flowchart that would resume the results obtained in CHCl3 solution for the chromophores taken into account in this chapter.

Figure II 18 .

 18 Figure II 18. Resuming flowchart of chromophores in CHCl3 solution studied in this chapter

  Illustration III 1. a) Antiparallel configuration of dipolar molecules represented by arrows, b) crystal packing of D1 ' c and c) crystal packing of D1 ''

Scheme III 2 .

 2 Scheme III 2. Schematic representation of the exciton band energy diagram for a molecular dimer with a) parallel transition dipoles and b) in-line transition dipoles. Where |g› and |e› are the ground and the excited states, ↔ represent the dipolar moment of the molecule and M is the transition moment of the dimer. Revised from Ref. 2

  Scheme III 3. Schematic representation of the exciton band energy diagram for a molecular dimer considering the angle θ between the single transition moments, where |g› and |e› are the ground and the excited states

Figure III 1 ,

 1 three chromophores of the same family bearing the same para-substituent but different acceptor groups (D2 ' b, D2 ' c and D2 ' d) are compared in terms of size distribution.

Figure III 1 .

 1 Figure III 1. Comparison between size distribution of nanoparticles prepared with chromophores D2 ' b (dark yellow), D2 ' c (orange) and D2 ' d (red)

Figure III 2 .

 2 Figure III 2. TEM images of a) D2 ' b, b) D2 ' c, c) D2 ' d

Figure III 3 . 3 . 1 .

 331 Figure III 3. Lambert-Beer dependency of nanoparticles prepared with chromophores of the series a) D2 ' a-d and b) D3 ' a-d

later in the chapter (Paragraph 4 :

 4 Molecular Confinement).

Figure III 4 .InFigure III 4

 44 Figure III 4. Comparison between the one-photon absorption spectra measured for the different members of the family a) D1 ' ; b) D3 ' and c) D3 '' upon nano-confinement.

Figure III 5 .

 5 Figure III 5. Comparison between normalized absorption (solid line) and emission (dashed line) spectra measured for the FONs prepared with the family a) D1 ' ; b) D3 ' and c) D3 '' (consistent color code between the figures of the paragraph)

  behavior observed in FONs parallels the one observed in solution, all the dipolar chromophores confined in nano-environment show a structured 2PA band in which λmax 2PA ≈ 2 λmax 1PA indicating that the transition is allowed in both processes (see Figure III 6).

Figure III 6 .

 6 Figure III 6. Superimposition between one-photon (dashed line) and two-photon (colored point and solid line) absorption spectra of chromophores a) D1 ' b; b) D3 ' c and c) D3 '' d in confined environment.

Figure III 7 . 2 .

 72 Figure III 7. Comparison between the two-photon absorption spectra measured for the different members of the family a) D1 ' ; b) D3 ' and c) D3 '' in FONs in water.

Figure III 8 .

 8 Figure III 8. Comparison between the one-photon absorption spectra measured for the different members of the series a) D1 ' -D3 ' c; b) D1 '' -D3 '' d in confined environment.

as well as in Figure III 9 ,

 9 FONs prepared with chromophores D3 ' c and D3 '' d are blue-shifted compared with the respective D1. This interesting behavior indicates that the tertbutylphenyl group within the nanoparticle does not work as donor, as observed in solution, but only as hindering group limiting interchromophoric interactions. Moreover, this property induces a slight enhancement of the fluorescence quantum yield, underlying our initial idea to prevent the π-π interaction in confined environment by adding bulky groups.

Figure III 9 .

 9 Figure III 9. Comparison between the normalized absorption (solid line) and emission (dashed line) spectra measured for chromophores a) D1 ' -D3 ' c; b) D1 '' -D3 '' d in nanoparticles (consistent color code between the figures of the paragraph)

6

  as example, also FONs displayed in this paragraph show that the position of the 2PA maximum is comparable or slightly red-shifted compared with twice λmax 1PA indicating that the occurring transition is symmetrically allowed upon both excitation one-photon and two-photon.Contrarily to what was observed for solution, the chromophores in FONs display a structured band but the intensity does not depend on the strength on the electron-releasing character (cf. Figure II 16). The trend of the series D ' c in FONs follows the expectation; the compounds display a hyperchromic effect passing from -Br to -H and to -tert-butylphenyl. On the other hand, in series D '' d, the FONs prepared with chromophore D2 '' d show the highest two-photon response, indicating an arrangement of the molecules within the nanoparticles that promote the 2PA enhancement.

Figure III 10 .

 10 Figure III 10. Comparison between the two-photon absorption spectra measured for the different chromophores bearing the same acceptor moiety a) c (cyanoester); b) d (dicyanovinyl) in FONs.

  and displayed in Figure III 11a, the FONs prepared with a more rigid system (D1 ' a → D1 '' a) show a hypsochromic shift in the ICT band associated with a slight a) b)enhancement of the molar extinction coefficient. Strikingly the same behavior is observable also by adding a fluorene core between two thiophenes, this structural change promotes, in FONs prepared with D1 ''' a, a marked enhancement of the molar extinction coefficient with a slight blue shift of the absorption band.A parallel behavior can be observed in emission. The substitution of a bis-thiophene as conjugated bridge with both thienothiophene and bis-thiophene-fluorene induces a blue shift of the band. As could be expected, in FONs the close interaction between neighbor molecules leads to a decreasing of the fluorescence quantum yield, and as it is reported in

Figure III 11

 11 Figure III 11. a) Comparison between the one-photon absorption spectra measured for the chromophores D1 ' -D1 ''' a in confined environment b) Normalized absorption and emission spectra measured for the chromophores D1 ' -D1 ''' a in FONs in water.

Figure III 12 .Figure III 13 .

 1213 Figure III 12. Superimposition between one-photon (dashed line) and two-photon (colored point and solid line) absorption spectra of chromophores a) D1 ' a; b) D1 ''' a; c) D1 ''' a in CHCl3 solution and d) comparison between the two-photon absorption spectra measured for the different members of the series D1a.

  absorption band associated with a bathochromic shift (or comparable emission i.e. D3 ' c, D3 ' d and D3 '' e) of the emission band, with respect to the measurement of the molecules in CHCl3 solution. This behavior suggests that the dye part of the bulk in the nanoparticle has a more polar environment compared with the chromophore in chloroform solution.

Figure III 14 .

 14 Figure III 14. Comparison between the one-photon absorption spectra measured in both CHCl3 solution (green line) and FONs (red line) for chromophores a) D1 ' c, b) D3 ' d, c) D2 '' d and d) D1 ''' a

  process (see Figure III 15a/d). On the other hand, the molecular arrangements as well as the interchromophoric interactions in FONs prepared with dye D3 ' d can lead to an hyperchromic effect in the 2PA response (Figure III 15b) or can induce a modification in the vibronic structure as displayed for chromophore D2 '' d in Figure III 15c.

Figure III 15 .

 15 Figure III 15. Comparison between the two-photon absorption spectra measured in both CHCl3 solution (green line) and FONs (red line) for chromophores a) D1 ' c, b) D3 ' d, c) D2 '' d and d) D1 ''' a

  , all FONs prepared show a very negative surface potential up to -85 mV. This indicates that the nanoaggregates could have a great colloidal stability; indeed, this is what was observed.

Figure III 16 .

 16 Figure III 16. Comparison between absorption (solid line) and emission (dashed line) spectra measured overtime for FONs prepared with chromophores: a) D1 ' a, b) D1 ' c, c) D3 ' a and d) D3 ' c

Figure III 17 .

 17 Figure III 17. Comparison between absorption (solid line) and emission (dashed line) spectra measured overtime for FONs prepared with chromophores: a) D1 ' a and b) D1 ''' a

Figure III 18 .

 18 Figure III 18. Absorption spectra measured overtime for chromophores D3 ' d and D3 '' e in DMSO solution adding one drop of water (a and c respectively) and confined in nanoparticles in water (b and d respectively)

Figure III 19 .

 19 Figure III 19. Resuming flowchart of chromophores in confined environment studied in this chapter

  Figure IV 1a-b one can see that DIp and DIIp 107 show a non-solvatochromic absorption band and a positive solvatochromism in emission, associated with a broadening of the band, as expected for classical push-pull chromophores; this behavior is explained by a marked ICT transition upon excitation that leads to an enhancement of the dipole moment. On the other hand, dyes DIp + and DIIp + (Figure IV 1c-d) display a negative solvatochromism in absorption with the exception of DCM in which is observable a red-shift of the

Figure IV 1 .

 1 Figure IV 1. Comparison between absorption (solid line) and emission (dashed line) measured in solvent of increasing polarity for chromophores a) DIp, b)DIIp, c) DIp + and d) DIIp +

Figure IV 2 .

 2 Figure IV 2. Comparison between absorption band of different chromophores studied in this chapter (consistent color code between the figures of the chapter)

  SDS in order to study their optical properties in this medium for further comparison with the molecule confined in nanoparticles.

Figure IV 4 .

 4 Figure IV 4. Comparison between absorption and emission measured for chromophore a) DIp + and b) DIq + in both SDS and THF solution; absorption and emission measured for the hemicyanine chromophores in SDS solution

,

  it was not possible to obtain ONPs with chromophore DIp + .

a)

  Estimated number of dyes subunit per ONPs based on their size As discussed earlier in the manuscript, since the number of dyes per nanoparticles depends strongly on the size of the object, we can have various orders of magnitude for FONs prepared with different chromophores. For example, as displayed in Figure IV 5, ONPs prepared with DIp display an average diameters of 104 nm with an estimated number of molecules per nanoparticles of 82.4 • 10 4 , while replacing the pyridine with the quinoline (DIq) one can observe an average diameter of 23 nm with a number of chromophores of 0.8 • 10 4 .

Figure IV 5 .

 5 Figure IV 5. Comparison between size distribution of nanoparticles prepared with chromophores DIp (dark yellow) and DIq (blue)

Figure IV 7 .(Figure IV 8 . 3 . 3 .Figure IV 9 .

 78339 Figure IV 7. Comparison between normalized absorption and emission of FONs prepared with hemicyanine dyes.

Chapter 3 ,

 3 Molecular confinement of a large number of chromophores generates strong intermolecular interactions that can induce the so-called aggregation caused quenching1 . As already discussed in to prevent this problem one can use the molecular engineering, designing molecules with hindering groups that limit the deleterious π-π stacking responsible of competitive non-radiative decay. The purpose is to prepare chromophores that are able to display AIE or AIEE and to do this we should take into account molecules that in solution are free to rotate while in confined environment some degrees of freedom are blocked in order to decrease the non-radiative decay rate, thus enhancing the emissive properties[2][3][4] .When one considers classical dipolar "push-pull" chromophores D-π-A, which usually display large intramolecular charge transfer (ICT) transition, a strong dipole-dipole interaction is expected hence an anti-parallel packing of molecules. This molecular arrangement can markedly affect both linear and non-linear optical properties 5 (cf. Chapter 3).Illustration V 1.Schematic representation of multidipolar chromophores studied in this chapter. The dipoles are represented by arrows2.1.1. Compound 1Bis(F)Single crystals of chromophore 1Bis(F) were grown by slow diffusion of MeOH in a supersaturated solution of dye in THF. The compound crystallizes in a centrosymmetric monoclinic P21/c space group.As it can be observed in Figure V 2a, the two dipoles connected by the fluorene core are in antiperiplanar conformation with a torsion angle between the two CHO acceptor groups of 172.5°. It is worthy to note the planarity distortion of the fluorene π bridge of 2° between the two phenyl rings.

  can observe a CH-π interaction between a butyl chain of a molecule and the fluorene of a chromophore forming the second layer (3.6 Å) while the distance between two fluorene cores is 7.5 Å, as displayed in Figure V 2b.

Figure V 2 2 .

 22 Figure V 2. a) Molecule 1Bis(F) in general position and b) view of the crystal packing along the b axis

Figure V 3 Figure V 4 . 3 . Photophysical characterization in organic solutions 3 . 1 .

 34331 Figure V 3. a) Molecule 2Bis(F) in general position and b) view of the crystal packing along the b axis

Figure V 5 .

 5 Figure V 5. Comparison between the absorption spectra measured for chromophores a) 1Mono, b) 1Bis(F), c) 1Bis(BP) and d) 1Tris

Figure V 6 .

 6 Figure V 6. Comparison between the absorption spectra measured for chromophores a) 1Mono, b) 1Bis(F), c) 1Bis(BP) and d) 1Tris

,

  chromophores with CHO as acceptor group display an intense absorption band, associated with a large molar extinction coefficient (up to 9.5 • 10 4 M -1 cm -1 ), that lies in the near UV spectral region. This band can be ascribed to an intramolecular charge transfer transition. As displayed in Figure V 7a, an increase of the number of dipolar subunits within the molecule induces a marked linear hyperchromic effect (see Figure V 7b), which is consistent with an additive effect. A slight bathochromic shift is observable from chromophores 1Mono going to 1Tris and 1Bis(F), this interesting effect will be discussed later in the chapter (Paragraph 6: Articulated dipoles: an essential state model description). Moreover, a hypsochromic shift associated with an hypochromic effect is observable by tuning the π-connector between the dipoles passing from F to BP, thanks to the fluorene that slight by increases the electron-releasing character, therefore enhancing the dipolar moment.

Figure V 7 .

 7 Figure V 7. Comparison between one-photon absorption spectra measured for a) 1Mono, 1Bis(F) and 1Tris, c) 1Bis and 2Bis (F and BP), b) linear dependency of the hyperchromic effect increasing the number of dipolar subunits within the molecule (consistent color code between the Figures in this chapter)

  Figures in this chapter)

  Comparing chromophores bearing CHO as acceptor group one can see in Figure V 8a that 1Mono, 1Bis(BP) and 1Tris display a good superimposition of the emission band, while 1Bis(F)

Figure V 8 .

 8 Figure V 8. Comparison between normalized absorption and emission spectra measured for a) 1Mono, 1Bis(F-BP) and 1Tris and b) 1Bis and 2Bis (F and BP)

3 .

 3 Anisotropy measurement in vitrified Me-THFTo complete the linear photophysical characterization, excitation anisotropy of chromophores 1Mono, 1Bis(F) and 1Tris was measured in vitrified Me-THF in order to study the ways of depolarization that the three dyes can follow.

Figure V 9 .

 9 Figure V 9. Excitation anisotropy (red line) overlapped with the excitation profile (black line) measured for chromophores a) 1Mono, b) 1Bis(F) and c) 1Tris in Me-THF at 77 K

Figure V 10 .Figure V 11 .

 1011 Figure V 10. Comparison between two-photon absorption spectra of a) 1Mono, 1Bis(F) and 1Tris, b) 1Bis(F-BP)

Figure V 12 .

 12 Figure V 12. TEM and AFM images of FONs prepared with the articulated dipoles presented in the chapter

Figure V 13 .

 13 Figure V 13. Comparison between one-photon absorption spectra measured for a) 1Mono, 1Bis(F) and 1Tris in FONs in water and b) linear dependency of the hyperchromic effect increasing the number of dipolar sub-units within the molecule

Figure V 14 .

 14 Figure V 14. Comparison between normalized absorption and emission spectra of chromophores 1Bis and 2Bis

Figure V 15 .

 15 Figure V 15. Comparison between the absorption spectra measured in THF solution and FONs in water for all chromophores described in this chapter

Figure V 16 .

 16 Figure V 16. Overlap between 1PA and 2PA spectra measured on FONs in water prepared with chromophores a) 1Bis(BP) and b) 2Bis(BP)

Figure V 17 .

 17 Figure V 17. Comparison between the two-photon absorption spectra measured in THF solution and FONs in water for chromophores a) 1Mono, b) 1Tris, c) 1Bis(F) and d) 1Bis(BP).

Figure V 18 ,Figure V 18 .

 1818 Figure V 18. Overtime monitoring of absorption and emission spectra of FONs prepared with the articulated dipoles

  a) the values of one-photon and two-photon brightness are reported taking into account the concentration of nanoparticles Both uptake and fluorescence properties of the nanoaggregates in bio-environment (approximate 20 pM concentration of FONs) were evaluated after 8 and 20 hours of incubation by two-photon fluorescence microscopy. Strong fluorescence signal was observed after 8h of incubation, particularly for the green FONs, the red ones appeared less intense. This is in agreement with the previously determined photophysical properties of the FONs: two-photon brightness of 2.6 10 6 GM for 1Bis(BP) and 0.9 10 6 GM for 2Bis(BP) in confined environment. As it is displayed in Figure V 19, both probes are broadly distributed in the cytosol, suggesting a non-specific internalization process.

Figure V 19 .

 19 Figure V 19. Two-photon fluorescence images of COS 7 cells incubated for 20h with both FONs 1Bis(BP) and 2Bis(BP); imaging conditions for a) 1Bis(BP): λexc = 750nm, λem = 450-550 nm and for c) 2Bis(BP): λexc = 900nm, λem = 600-750 nm. b) display the co-localization of both probes.

  investigated their biocompatibility. The cytotoxic effect was evaluated by MTT assay, after 24h of incubation with the COS 7 cells, as displayed in Figure V 20. None of the FONs proved to be toxic at the concentration used for bioimaging (approximately 20 pM), the viability of the cells being of about 100% in all conditions. The safety of these FONs as well as their photostability and high brightness indicate the great potential of these FONs in biomedical applications, including imaging and sensing.

  dipolar dye with the two-state model, then to consider the other chromophores 1Bis(F) and 1Tris, displayed in Figure V 21, as a combination of two or three dipolar units interacting via electrostatic interactions.

Figure V 21 .

 21 Figure V 21. Target molecules used for calculation, violet 1Mono, green 1Bis(F) and magenta 1Tris

Figure V 22 .Figure V 23 .

 2223 Figure V 22. Comparison between both absorption and emission spectra measured experimentally (dashed line) in solvent with different polarity and calculated with the essential state model (solid line) for chromophores 1Mono and 1Bis(F)

Figure V 24 . 1 . 2 .

 2412 Figure V 24. Comparison between absorption and emission spectra measured experimentally (dashed line) in solvent with different polarity and calculated with the essential state model (solid line) for chromophores 1Mono, 1Bis(F) and 1Tris

Figure V 25 . 6 . 3 .

 2563 Figure V 25. Comparison between both 1PA and 2PA spectra measured experimentally and calculated with the essential state model, using THF as common solvent, for chromophores 1Mono, 1Bis(F) and 1Tris

Figure V 26 .

 26 Figure V 26. Comparison between the experimental (dashed line) and the calculated (solid line) excitation profile (black) and anisotropy (red)

Figure V 27 .

 27 Figure V 27. Resuming flowchart of articulated dipoles confined in nanoparticles

  VI 2. Energy level diagrams for general centrosymmetric and noncentrosymmetric chromophores (where |𝐠〉 and |𝐞〉 are the ground and the excited state respectively, and the subscripted g and u are gerade and ungerade symmetry respectively).

Figure VI 1 .

 1 Figure VI 1. Quadrupolar D-π-A-π-D chromophores studied in this chapter

Figure VI 2

 2 Figure VI 2. a) QfvB1 in general position, b) dimer generated by the inversion center and c) view of the crystal packing

1 .Figure VI 3 .

 13 Figure VI 3. Normalized absorption and emission spectra measured in solvent of increased polarity to study the solvatochromic behavior of the quadrupolar chromophores discussed in this chapter (in the normalized absorption spectra the high energy band ascribed to π-π * transition was cut out for clarity)

Figure VI 4

 4 fluorene with a conjugated linker (QfB1 → QfvB1, QfeB1 and QftB1) leads to a bathochromic shift of the ICT band indicating the extension of electronic conjugation. As expected8 the change of a triple bond by a double bond (QfeB1 → QfvB1) induces a shift towards low energies linked to a marked hyperchromic effect, in line with an increase of the conjugated path. Interestingly, the replacement of a double bond by a thienyl unit leads to a bathochromic shift associated, unexpectedly, with a decrease of the molar extinction coefficient. The low aromaticity of the thienyl moiety decreases the gap between the ground and the excited state probably lowering the ICT transition.

  elongating the π-conjugated bridge, from QfB1 to the other members of the class, it can be observed a bathochromic shift of the emission band, but in Figure IV 4b is displayed that the replacement of the ethynyl linker with the vinyl one induces an unexpected red-shift associated with a marked increase of the fluorescence quantum yield (from 0.18 to 0.59). Strikingly, the obtained chromophores, especially QfvB1 and QftB1, display good Φf in the red visible region.

Figure VI 4 .

 4 Figure VI 4. Comparison between a) one-photon absorption spectra and b) normalized absorption and emission spectra measured for the quadrupoles with different π bridge in THF solution (in the normalized absorption was cut out the higher energetic band ascribed to π-π * transition for clarity) (consistent color code between the figures of the chapter)

Figure VI 5 .

 5 Figure VI 5. Comparison between a) one-photon absorption spectra and b) normalized absorption and emission spectra measured for the quadrupoles increase the number of benzothiadiazole in THF solution (in the normalized absorption was cut out the higher energetic band ascribed to π-π * transition for clarity) (consistent color code between the figures of the chapter)

  From the comparison between the 2PA spectra displayed in Figure VI 6e one can clearly see that the elongation of the conjugation with an additional linker (QfB1 → QfvB1, QfeB1 and QftB1) induced a slight red-shift of the ICT band associated a marked hyperchromic effect, paralleling what was observed upon one-photon excitation. It is interesting to underline that the addition of ethynyl, vinyl or thienyl moiety in the conjugated bridge induces also the structuration of the band that cannot be observed for chromophore QfB1. The substitution of the ethynyl linker with the vinyl one leads to an increase of the two-photon cross section linked to the enhancement of the conjugated character of the bridge. Moreover, the replacement of the double bond with the thienyl group induces a marked bathochromic shift of the λmax 2PA .

Figure VI 6

 6 Figure VI 6. a-d) Comparison between 1PA (dashed line) and 2PA (colored point and solid line) spectra measured for chromophores by tuning the π bridge dissolved in THF solution, e) Comparison of 2PA spectra of the chromophores of the family.

Figure VI 8 .

 8 Figure VI 8. Size distribution (left panel) and TEM image (right panel) of FONs prepared with a) QfB1, b) QfvB1,c) QfeB1,d) QftB1 and e) QfeB2.

Figure VI 9 .

 9 Figure VI 9. Comparison between one-photon absorption spectra and normalized emission measured for chromophores a) QfB1, b) QfvB1,c) QfeB1,d) QftB1 in both THF solution and FONs in water.

Figure VI 10 .

 10 Figure VI 10. Normalized emission spectra of the quadrupolar chromophores upon nanoaggregation

Figure VI 11 .

 11 Figure VI 11. Comparison between the 2PA spectra measured for the quadrupoles in both THF solution (green colored point and solid line) and FONs in water (colored point consistent color code between the figures)

2 .

 2 Figure VI 9c respectively).

Figure VI 12

 12 Figure VI 12. a) Comparison between absorption and normalized emission spectra of the FONs prepared with QfeB1-2; b) Comparison between one-photon absorption spectra and normalized emission measured for chromophore QfeB2 in both THF solution and FONs in water

  noticed in Figure VI 13b that the addition of a second BTDA in the core induces a weak hypochromic effect does not affecting significantly the absorption band.From a direct comparison between the two-photon absorption spectra measured for the chromophore QfeB2 in both THF solution and FONs in water displayed in Figure VI 13c, one can see that both spectra show a structured band and it is interesting to highlight that the relative intensities of bands is different, specifically the most intense band measured in FONs correspond to the less intense band measured for the dye in THF, causing a bathochromic shift of the λmax 2PA . Moreover, It is worthy to underline that with passing from molecular solution to nanoconfinement a pronounced shoulder appears at low energies, suggesting an excitonic coupling upon aggregation.

Figure VI 13

 13 Figure VI 13. a) Overlap between one-photon absorption (dashed line) and two-photon absorption (solid line and colored points) measured for chromophore QfeB2 in confined environment; comparison between 2PA spectra measured on FONs QfeB1-2 and c) comparison between normalized 2PA spectra measured for QfeB2 in both THF solution and FONs in water

Figure VI 14 .

 14 photon microscopy (d-f) using as probes the FONs prepared with QfvB1 and QfeB2 respectively,

Figure VI 16 .

 16 Figure VI 16. Viability of COS 7 cells after 24h and 48h of incubation with FONs prepared with a) QfvB1 and b) QfeB2

  This manuscript can be inserted in the wide topic, very active nowadays, towards the achievement of new bright probes suitable for imaging in biological media. I focused my work in the design,the synthesis and the photophysical studies of new chromophores for the further preparation of full organic fluorescent organic nanoparticles (FONs). The work presented in this Thesis show four different classes of new chromophores with tunable photophysical properties, specifically with tunable emission that cover the visible range towards NIR region: neutral dipoles (Chapter II and III), charged dipoles (Chapter IV), articulated dipoles (Chapter V) and quadrupoles (Chapter VI).



  AFM = atomic force microscopy  BTDA = benzothiadiazole  CHO = aldehyde  DAPI = 4',6'-diethylamidino-2-phenylindole  DETB = 1,3-Diethyl-2-thiobarbiturate  FONs = fluorescent organic nanoparticles  FWHM = full width at half maximum  IC = internal conversion  ICT = intramolecular charge transfer  ISC = intersystem crossing  NIR = near infrared  NLO = non-linear optics  ONPs = organic nanoparticles  TEM = transmission electron microscopy  TPEF = two-photon excited fluorescence  2PA = two-photon absorption  2PM = two-photon microscopyTEM was carried out using a HITACHI H7650. The copper grid coated with a carbon membrane was pretreated using the Glow discharge technique to yield positively charged hydrophilic carbon surface to allow stronger interaction between the sample and the grid itself and thus easier imaging. One droplet of the dilute aqueous suspension was deposited on the grid and the excess liquid was dried off with a paper. A staining procedure using uranyl acetate was then used to enhance the contrast. AFM analysis, 20 µl of suspension of FONs was deposited onto freshly cleaved mica disks and left to dry at room temperature. A Bruker's Dimension Icon  , Atomic Force Microscope operated in peak force mode, available at SIV-ISM (Bordeaux) was used. ScanAsyst-Air (Bruker) probes were employed, with a set point of 500 pN and the scan rate 0.250 Hz. The images were processed with NanoScope Analysis 1.5 program. Zeta-potential analysis was performed with the same instrument, 20 measurements were realized for each sample according to a predefined operating procedure. The global concentration of the dye in the FONs suspension was determined by taking an aliquot of the suspension, which was further lyophilized then dissolved in the same volume of CHCl3. The concentration was then derived from the absorbance and the value of the molar extinction coefficient in CHCl3 by using the Lambert-Beer law.1.4. Photophysical characterizationAll photophysical studies have been performed with freshly prepared air-equilibrated solutions at room temperature (298 K). UV/Vis absorption spectra were recorded on a Jasco V-670 spectrophotometer. Steady-state measurements were carried out on a Fluoromax-4 spectrofluorometer and time resolved fluorescence measurements were carried out on a Fluorolog spectrofluorometer. The emission spectra were corrected for the wavelength-sensitivity of the detection unit, and they were obtained under excitation at the wavelength of the absorption maximum. Fluorescence quantum yields of dilute dye solutions and of the FONs suspensions were measured according to literature procedures 2,3 using DCM (Φ = 0.437 in EtOH, λexc = 443 nm), Nile Blue (Φ = 0.27 in EtOH, λexc = 200-610 nm range), Cresyl Violet (Φ = 0.54 in MeOH, λexc = 570 nm), Indocyanine Green (Φ = 0.11 in DMSO λexc = 678 nm), Rhodamine-6G (Φ = 0.94 in EtOH, λexc = 488 nm) or Fluorescein in NaOH 0.1 M (Φ = 0.9 in NaOH 0.1 M, λexc = 474 nm) depending on the emission range4 . The reported fluorescence quantum yield values obtained via this method are within ± 0.005. In Eq. 1 is displayed the equation used to calculate the Φf of the sample taking into account the refraction index n, the absorbance A and the integral of the emission I.Fluorescence decays were measured in a time-correlated single photon counting (TCSPC) configuration, under excitation from a NanoLED (370 nm, 455 nm or 570 nm). The instrument response was determined by measuring the light scattered by a Ludox ® suspension. The lifetime values were obtained from the deconvolution fit analysis of the decays profiles; the quality of the fits was judged by the reduced χ² value (0.9< χ² < 1.1). The reported lifetimes are within ± 0.1 ns.Anisotropy measurements were carried out on a Fluorolog spectrofluorometer using triacetin at 20°C (293.15 K) or on a Fluoromax-3 using 2-MeTHF at -203.15°C (70 K) Two-photon absorption cross sections (σ2) were determined from the two-photon excited fluorescence (TPEF) cross sections (σ2Φ) and the fluorescence quantum yield (Φ). TPEF cross sections were measured relative to Fluorescein in 0.01 M aqueous NaOH in the 680-1000 nm spectral range and relative to Nile Red in DMSO in the 1000-1160 nm spectral range, using the method described by Xu and Webb 5 and the appropriate solvent-related refractive index corrections. The quadratic dependence of the fluorescence intensity on the excitation power was checked at all wavelengths. Measurements were conducted using an excitation source delivering fs pulses. This allows avoiding excited-state absorption during the pulse duration, a phenomenon, which has been shown to lead to overestimated two-photon absorption cross-section values. To scan the 680-1080 nm range, a Nd:YVO4-pumped Ti:Sapphire oscillator was used generating 140 fs pulses at a 80 MHz rate.

FTIR(

  ATR): ν (CHarom/HC=C) 3070 cm -1 ; ν (N(CH3)2) 2917 cm -1 ; ν (C≡N) 2198 cm -1 ; ν (C=O) 1637 cm -1 . HRMS (m/z) [M]calcd for C32H25N3OS2: 531.14390, found: 531.14600.Methyl-2-cyano-3-[5'-[4-(diphenylamino)phenyl][2,2'-bithiophen]-5-yl]acryate (D1 ' c):Following the general procedure using D1 ' a as aldehyde and methylcyanoacetate as α-acid derivatives to yield compound D1 ' c as a bright red solid (yield = 80%).

FTIR(

  ATR): ν (CHarom/HC=C) 3080 cm -1 ; ν (O(CH3)) 2950 cm -1 ; ν (C≡N) 2210 cm -1 ; ν (C=O) 1724 cm -1 . HRMS (m/z) [M]calcd for C31H22N2O2S2: 518.11227, found: 518.11021.

FTIR

  (ATR): ν (CHarom/HC=C) 3025 cm -1 ; ν (O(CH3)) 2948 cm -1 ; ν (C≡N) 2214 cm -1 ; ν (C=O) 1721 cm -1 .HRMS (m/z) [M] calcd for C31H20Br2N2O2S2: 673.93329, found: 673.93211. Following the general procedure using D2 ' a as aldehyde and malononitrile as α-acid derivatives to yield compound D2 ' d as a deep purple solid (yield = 96%).

1 H-

 1 NMR (300 MHz, CDCl3): 7.89 (s, 1H); 7.72 (d, 1H, J = 8Hz); 7.51 (d, 1H, J = 8Hz); 3.86 (s, 1H) ppm. 2,7-diiodo-9,9-diisopentyl 9H fluorene (3): In a solution of KOH (0.189 mol in 15 mL of water)

1 H- 2 , 2 '-( 9 , 9 -

 12299 NMR (300 MHz, CDCl3): 7.67 (d, J = 7.9 Hz, 4H); 7.42 (d, J = 7.9 Hz, 2H); 1.92 (m, 4H); 1.31 (m, 2H); 0.72 (d, J = 6.6 Hz, 12H); 0.49 (m, 4H) ppm. diisopentyl 9H fluorene-2,7 diyl) dithiophene (4): In a Schlenk flask were insert Mg (8.14 mmol, 4.55 eq.), one crystal of iodine and 32 mL of dry THF, under Ar. While stirring the temperature was decreased at -40°C, at the mixture were added dropwise bromothiophene (8.14 mmol, 4.55 eq.) and the reaction was left at -40°C for 1h30. In the meanwhile in another Schlenk flask were introduced 3 (1.79 mmol, 1 eq.) and Pd(dppf)Cl2 (0.054 mmol, 0.03 eq.). After 1h30 the solution of 5-(7-(4-(diphenylamino)phenyl)thiophene-2-yl)-9,9-diisopentyl 9H fluorene-2-yl)thiophene-2-carbaldehyde (D1 ''' a): 6 (0.153 mmol; 1 eq.), 4-(diphenylamino)phenylboronic acid (0.184 mmol;

1 H-

 1 NMR (300 MHz, CDCl3): 9.93 (s, 1H); 7.79 (d, J = 4 Hz, 1H); 7.74 (m, 3H) 7.64 (m, 1H); 7.60 (m, 1H); 7.54 (d, J = 8.8 Hz, 2H); 7.50 (d, J = 3.8 Hz, 1H); 7.38 (d, J = 3.8 Hz, 1H); 7.32 (m, 4H); 7.26 (d, J = 3.8 Hz, 1H); 7.18 (m, 3H); 7.15 (m, 2H); 7.12 (m, 3H); 7.06 (m, 1H); 2.08 (m, 4H); 1.34 (m, 2H); 0.73 (d, J = 6.8 Hz, 12H); 0.58 (m, 4H) ppm.

  •

		𝑛 𝑆 2 𝑛 𝑅 2 •	𝐹 𝑆 𝐹 𝑅	•	𝑓 𝑅 (𝜆 𝑒𝑥𝑐 ) 𝑓 𝑆 (𝜆 𝑒𝑥𝑐 )
	with
	Eq I 6.	𝜆 2 𝐹 = ∫ 𝐼 𝑐 (𝜆 𝑒𝑥𝑐 , 𝜆 𝑒𝑚 )d𝜆 𝑒𝑚 𝜆 1

Table I

 I

1.

Photophysical properties of some endogenous chromophores

1,[START_REF] Lavis | Bright ideas for chemical biology[END_REF]

Cpd λmax 1PA [nm] εmax [M -1 cm -1 ] λmax em [nm] Φf εmaxΦf [M -1 cm -1 ]

	Tyrosine	275	1500	303	0.14	210
	Tryptophane	280	6300	348	0.13	820
	NADH	340	6200	435	0.019	120
	FMN a)	450	12200	530	0.25	3100
	a) Flavin mononucleotide

CHAPTER 2 -DIPOLAR CHROMOPHORES IN MOLECULAR SOLUTION

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cm -1 ] εmax [10 4 M -1 cm -1 ] λmax em [nm] Stokes shift [10 3 cm -1 ] Φf τ1/τ2 [ns] kr [10 9 s -1 ] knr [10 9 s -1 ]

  2.One-photon photophysical data measured in CHCl3 solution for the chromophores of the families D1

	λmax 1PA [nm] [10 3 D1 ' a FWHM Cpd 432 4.6	3.1	586	6.1	0.80	3.1	0.26	0.06
	D1 ' b	469	4.7	3.7	648	5.9	0.50	1.3 (0.2)/2.7 (0.8)	0.23	0.23
	D1 ' c	495	4.4	3.7	680	5.5	0.60	3.1	0.19	0.13
	D1 ' d	521	4.0	3.3	706	5.0	0.55	3.0	0.18	0.15
	D3 ' a	441	4.7	3.3	607	6.2	0.80	3.4	0.24	0.06
	D3 ' c	503	4.6	3.7	705	5.7	0.40	2.5	0.16	0.24
	D3 ' d	530	4.2	3.5	748	5.5	0.25	1.5	0.17	0.50
	D3 '' a	429	4.2	3.9	582	6.1	0.70	3.3	0.21	0.09
	D3 '' d	525	3.7	4.5	700	4.8	0.25	1.9	0.13	0.39
	D3 '' e	576	3.4	5.5	755	4.1	0.06	0.6	0.10	1.57

' a-d, D3 ' a-d, D3 '' a-e.

Table II

 II

	Cpd	λmax 1PA [nm]

3.

One-photon photophysical data measured in CHCl3 solution for the chromophores D1 ' -D3 ' c and D1 '' -D3 '' d.

εmax [10 4 M -1 cm -1 ] λmax em [nm] Stokes shift [10 3 cm -1 ]

						Φf	τ1 [ns]	kr [10 9 s -1 ]	knr [10 9 s -1 ]
	D1 ' c	495	3.7	680	5.5	0.60	3.1	0.19	0.13
	D2 ' c	483	3.9	652	5.4	0.75	2.8	0.27	0.09
	D3 ' c	503	3.7	705	5.7	0.40	2.5	0.16	0.24
	D1 '' d	511	4.2	652	4.2	0.47	2.6	0.18	0.20
	D2 '' d	495	5.4	629	4.3	0.31	1.5	0.21	0.46
	D3 '' d	525	4.5	700	4.8	0.34	1.9	0.18	0.35

Table II

 II

	4.	One-photon photophysical data measured in CHCl3 solution for
	the chromophores D1

' -D1 ''' a. Cpd λmax 1PA [nm] εmax [10 4 M -1 cm -1 ] λmax em [nm] Stokes shift [10 3 cm -1 ] Φf τ1 [ns] kr [10 9 s -1 ] knr [10 9 s -1 ]

	D1 ' a	432	3.1	586	6.1	0.80	3.1	0.26	0.06
	D1 '' a	417	3.9	534	5.2	0.84	2.8	0.30	0.06
	D1 ''' a	415	5.1	609	7.7	0.20	1.2	0.17	0.68

Table II

 II

	Cpd	2λmax 1PA [nm]	λmax 2PA [nm]	σ2 [GM]	λmax2 2PA [nm]	σ2 [GM]
	D1 ' a	864	910	302	850	246
	D1 ' b	938	940	277	890	273
	D1 ' c	990	1050	343	980	301
	D1 ' d	1042	1090	492	980	268
	D3 ' a	882	940	422	850	280
	D3 ' c	1006	1050	647	980	490
	D3 ' d	1060	1090	813	980	340
	D3 '' a	858	890	479	850	428
	D3 '' d	1050	1060	1402	980	813
	D3 '' e	1152	1120	2002	/	/

5. Two-photon photophysical data measured in CHCl3 solution for the chromophores of the families D1 ' a-d, D3 ' a-d, D3 '' a-e.

' a-d, D3 ' a-d, D3 '' a-e.

	a)				b)
			c)
	Table II 6.	Two-photon photophysical data measured in CHCl3 solution for
	2λmax 1PA the chromophores of the families D1 Cpd λmax 2PA σ2 λmax2 2PA [nm] [nm] [GM] [nm]	σ2 [GM]	λmax3 2PA [nm]	σ2 [GM]
	D3 ' a	990	1050	344	980	301	890	250
	D3 ' c	966	1030	254	960	280	890	262
	D3 ' d	1006	1050	647	980	490	890	451
	D3 '' a	1022	1050	1164	970	951	/	/
	D3 '' d	990	1050	778	960	933	/	/
	D3 '' e	1050	1060	1402	980	813	/	/

Table II

 II

		7.	Two-photon photophysical data measured in CHCl3 solution for
		the chromophores of the series D1a.
	Cpd	2λmax 1PA [nm]	λmax 2PA [nm]	σ2 [GM]	λmax2 2PA [nm]	σ2 [GM]	λmax3 2PA [nm]	σ2 [GM]
	D1 ' a	864	910	302	850	246	/	/
	D1 '' a	834	830	238	/	/	/	/
	D1 ''' a	828	890	467	830	526	720	814

  1 are gathered the relevant data concerning the morphological properties of the ONPs.

	Table III 1.	Morphological and structural data
	Cpd	dTEM [nm]	N a) [10 4 ]	ζ-potential [mV]
	D1 ' a	36	4.0	-40
	D1 ' b	27	1.2	-62
	D1 ' c	40	3.9	-61
	D1 ' d	41	4.5	-68
	D2 ' a	38	2.9	-73
	D2 ' b	66	13.1	-63
	D2 ' c	38	2.6	-69
	D2 ' d	21	0.4	-68
	D3 ' a	40	0.9	-81
	D3 ' c	32	1.3	-73
	D3 ' d	45	3.9	-75
	D1 '' a	42	5.7	-70
	D1 '' d	21	0.6	-74
	D1 '' e	30	1.4	-67
	D2 '' a	37	2.8	-85
	D2 '' d	39	3.0	-75
	D2 '' e	29	1.0	-75
	D3 '' a	27	0.9	-72
	D3 '' d	32	1.5	-79
	D3 '' e	32	1.2	-72
	D1 ''' a	34	1.7	-73

a) Estimated number of dyes subunits per ONPs based on their size

M -1 cm -1 ] λmax em [nm] Stokes shift [10 3 cm -1 ]

  Table III 2. Photophysical data measured for the chromophores of the series D1

	Cpd	λmax 1PA [nm]	εmax [10 4 Φf	τ1/τ2 [ns]	λmax 2PA [nm]	σ2max [GM]
	D1 ' a	422	2.3	597	6.9	0.07	1.1 (0.6) / 3.5 (0.4)	970
	D1 ' b	458	2.6	673	7.0	0.06	1.6 (0.7) / 4.2 (0.3)	890
	D1 ' c	481	2.5	721	6.9	0.02	1.4 (0.8) / 4.1 (0.2)	1070
	D1 ' d	513	3.6	770	6.5	0.01	1.5 (0.8) / 4.9 (0.2)	1110
	D3 ' a	441	3.1	587	5.6	0.12	1.3 (0.5) / 4.2 (0.5)	970
	D3 ' c	499	2.7	702	5.8	0.04	1.5 (0.4) / 3.8 (0.6)	1050
	D3 ' d	525	3.0	747	5.7	0.01	nd	1050	1150

' a-d, D3 ' a-d, D3 '' a-e in FONs in water.

Table III

 III

3. Photophysical data measured for the FONs prepared with chromophores D1 ' -D3

' c and D1 '' -D3 '' d. Cpd λmax 1PA [nm] εmax [10 4 M -1 cm -1 ] λmax em [nm] Stokes shift [10 3 cm -1 ]

	a)				b)
						Φf	τ1/τ2 [ns]	λmax 2PA [nm]	σ2max [GM]
	D1 ' c	481	2.5	721	6.9	0.02	1.4 (0.8)/4.1 (0.2)	1070	204
	D2 ' c	476	2.7	688	6.5	0.04	1.4 (0.8)/3.8 (0.2)	1060	141
	D3 ' c	499	2.7	702	5.8	0.04	1.5 (0.4)/3.8 (0.6)	1050	336
	D1 '' d	476	3.5	719	7.1	0.01	1.5 (0.6)/4.0 (0.4)	1070	300
	D2 '' d	478	6.1	711	6.9	0.01	1.1 (0.7)/3.5 (0.3)	1060	921
	D3 '' d	507	5.0	714	5.7	0.02	1.5 (0.5)/4.6 (0.5)	1080	726

Table III

 III

		5.	Luminescence properties including one-photon and two-photon
		brightness of FONs in water
	Cpd	λmax 1PA [nm]	λmax em [nm]	FWHM em [10 3 cm -1 ]	Φf	εmaxΦf a) [10 8 M -1 cm -1 ]	λmax 2PA [10 4 ]	σ2Φf a) [10 6 GM]
	D1 ' a	422	597	3.0	0.07	0.7	970	0.3
	D1 ' b	458	673	2.8	0.06	0.2	890	0.1
	D1 ' c	481	721	2.6	0.02	0.2	1070	0.2
	D1 ' d	513	770	2.4	0.01	0.2	1110	0.2
	D2 ' a	424	575	3.1	0.14	1.1	970	0.4
	D2 ' b	451	653	3.0	0.06	2.5	900	1.4
	D2 ' c	476	688	2.7	0.04	0.3	1060	0.3
	D2 ' d	500	732	2.6	0.02	0.03	1060	0.02
	D3 ' a	441	587	3.1	0.12	0.3	970	0.8
	D3 ' c	499	702	2.6	0.04	0.1	1050	0.2
	D3 ' d	525	747	2.5	0.01	0.1	1050	0.2
	D1 '' a	410	557	3.1	0.05	1.0	910	1.1
	D1 '' d	476	719	2.5	0.01	0.02	1070	0.02
	D1 '' e	517	754	2.5	0.004	0.03	/	/
	D2 '' a	407	541	3.0	0.05	0.5	890	0.8
	D2 '' d	478	711	2.5	0.01	0.2	1060	0.3
	D2 '' e	509	752	2.6	0.007	0.04	/	/
	D3 '' a	424	570	3.6	0.06	0.4	910	0.2
	D3 '' d	507	714	2.5	0.02	0.4	1080	0.5
	D3 '' e	541	749	2.5	0.005	0.02	/	/
	D1 ''' a	409	557	3.7	0.035	0.3	730	0.4
	a) the values of one-photon and two-photon brightness are reported taking into account the concentration of nanoparticles

Table IV

 IV

	2.	Comparison between photophysical data of chromophores in
	THF and SDS solution
	Cpd	λmax 1PA [nm]	εmax [10 4 M -1 cm -1 ]	λmax em [nm]	Φf	τ1 [ns]
	DIp + (THF)	500	3.1	745	0.03	0.48 (0.8)/2.17 (0.2)
	DIp + (SDS)	477	3.9	650	0.14	0.90 (0.9)/2.04 (0.1)
	DIq + (THF)	555	2.7	835	0.004	/
	DIq + (SDS)	530	1.8	710	0.07	0.80
	DIIp + (THF)	506	3.3	779	0.005	0.51 (0.8)/3.28 (0.2)
	DIIp + (SDS)	484	1.2	680	0.11	0.95
	DIIq + (THF)	557	3.1	/	/	/
	DIIq + (SDS)	534	2.4	754	0.03	0.51

Table IV

 IV

		4.	Photophysical data measured for the hemicyanine
		chromophores upon nanoaggregation
	Cpd	λmax 1PA [nm]

εmax Chrom b) [10 4 M -1 cm -1 ] εmax FONs c) [10 9 M -1 cm -1 ] λmax em [nm] Φf εmax FONs Φf [10 6 M -1 cm -1 ]

	DIp +	/	/	/	/	/	/
	DIq +	553	4.8	11	830	0.002	22.0
	DIIp +	468	3.2	2.1	771	0.002	4.2
	DIIq +	545	3.7	1.2	889	0.0005 a)	0.6
	a) Because of the low fluorescence quantum yield this data is an average between 5 independent measurements, b)
	estimated value taking into account the total concentration of chromophores in nanoparticles, c) estimated value taking into
	account the concentration of FONs

Table V

 V

		1.	One-photon photophysical data for chromophores in THF
	solution
	Cpd	λmax 1PA [nm]	εmax [10 4 M -1 cm -1 ]	λmax em [nm]	Φf	τ1 [ns]	kr [10 9 s -1 ]	knr [10 9 s -1 ]
	1Mono	367	2.6	503	0.30	5.3	0.06	0.13
	1Bis(F)	382	5.6	530	0.11	2.2	0.05	0.40
	2Bis(F)	453	7.8	670	<0.01	/	/	/
	1Bis(BP)	369	4.8	511	0.16	2.9	0.06	0.29
	2Bis(BP)	448	6.8	653	<0.01	/	/	/
	1Tris	376	9.5	508	0.39	5.3	0.07	0.12

Table V

 V

	2.	Two-photon photophysical data for chromophores in THF
	solution
		Cpd	2λmax 1PA [nm]	λmax 2PA [nm]	σ2max [GM]
		1Mono	734	750	103
		1Bis(F)	764	740	144
		1Bis(BP)	738	740	250
		1Tris	752	740	225

  .

	c)			d)
	Table V 3.	Morphological and structural data
	Cpd	dTEM [nm]	dAFM [nm]	dTEM/AFM a) [nm]	N [10 4 ]	ζ-potential [mV]
	1Mono	73.5	66.5	70	22.8	-54
	1Bis(F)	33	27	30	1.1	-68
	2Bis(F)	34	36	35	1.5	-57
	1Bis(BP)	60	50	55	9.6	-66
	2Bis(BP)	36	48	42	3.6	-65
	1Tris	44.5	51.5	48	2.8	-64
	a) average diameter between TEM and AFM

Table V

 V

	4.	One-photon photophysical data for chromophores confined in
	FONs in water
	Cpd	λmax 1PA [nm]	εmax [10 4 M -1 cm -1 ]	λmax em [nm]	Φf	τ1/τ2 [ns]
	1Mono	370	2.3	488	0.24	1.9 (0.4)/6.5 (0.6)
	1Bis(F)	378	4.8	512	0.13	1.5 (0.4)/4.8 (0.6)
	2Bis(F)	453	7.4	656	0.02	0.7 (0.9)/2.8 (0.1)
	1Bis(BP)	370	3.7	511	0.10	1.7 (0.4)/5.5 (0.6)
	2Bis(BP)	444	7.0	643	0.03	0.9 (0.8)/2.9 (0.2)
	1Tris	375	8.3	499	0.20	1.6 (0.4)/5.7 (0.6)

Table V

 V

	5.	Molecular two-photon response of chromophores confined in
	FONs in water
		Cpd	2λmax 1PA [nm]	λmax 2PA [nm]	σ2 max [GM]
		1Mono	740	750	87
		1Bis(F)	756	740	157
		1Bis(BP)	740	750	222
		2Bis(F)	906	940	727
		2Bis(BP)	888	900	816
		1Tris	750	750	130

Table V

 V

		6.	Luminescence properties including one-photon and two-photon
		brightness of FONs in water
	Cpd	λmax 1PA [nm]	λmax em [nm]	FWHM em [10 3 cm -1 ]	Φf	εmaxΦf a) [10 8 M -1 cm -1 ]	λmax 2PA [10 4 ]	σ2Φf a) [10 6 GM]
	1Mono	370	488	3.6	0.24	14.8	750	5.0
	1Bis(F)	378	512	3.8	0.13	0.7	740	0.2
	1Bis(BP)	370	511	3.9	0.10	6.1	750	2.6
	2Bis(F)	453	656	3.0	0.02	0.3	900	0.4
	2Bis(BP)	444	643	3.0	0.03	0.9	900	0.6
	1Tris	375	499	3.7	0.20	5.5	750	0.7

  7.

	Table V 7.	Essential state model parameters
		Description	Parameter
	Electronic part	Half of the energy gap between the neutral state and the zwitterionic state expressed in eV Mixing element of the Hamiltonian expressed in eV	z0 √2 t
	Vibrational part	Frequency of the vibration expressed in eV Related to the vibrational relaxation energy, is in eV called electron-phonon coupling constant expressed	ωvib g
	Solvent contribution	Solvation relaxation energy expressed in eV	εor
		Linewidth of the vibronic transition expressed in eV	γel
		Permanent dipole moment of the zwitterionic state expressed in D	μ0

Table V

 V

	8.	Parameters used to reproduce the experimental data with good
	agreement
	Parameters	1Mono	1Bis(F)
		z0	1.585	1.495
		√2 t	0.8	0.8
		ωvib	0.2	0.2
		g	0.215	0.215
			0.26 (cyclohex)	0.26 (cyclohex)
		εor	0.585 (THF)	0.585 (THF)
			0.855 (DCM)	0.855 (DCM)
		γel	0.04	0.04
		μ0	27.8	27.8

  Table V 9, it was necessary to change the value of z0 when going to dimer and trimer species, in order to reproduce the slight experimental shift observed in the absorption band.

	Table V 9.	Parameters used to reproduce the experimental data with good
	agreement
	Parameters	1Mono	1Bis(F)	1Tris
	z0	1.55	1.485	1.505
	√2 t	0.7	0.7	0.7
	ωvib	0.2	0.2	0.2
	g	0.21	0.21	0.21
		0.305 (cyclohex)	0.305 (cyclohex)	0.305 (cyclohex)
	εor	0.615 (THF)	0.615 (THF)	0.615 (THF)
		0.835 (DCM)	0.835 (DCM)	0.835 (DCM)
	γel	0.15	0.15	0.15
	μ0	29.15	29.15	29.15

Table VI

 VI

	1.	Linear photophysical relevant data
	Cpd	λmax 1PA [nm]	εmax [10 4 M -1 cm -1 ]	λmax em [nm]	Φf	τ1 [ns]
	QfB1	450	2.5	628	0.64	5.15
	QfvB1	505	5.9	644	0.59	3.61
	QfeB1	474	3.9	688	0.18	2.20
	QftB1	528	4.5	675	0.45	3.88

Table VI

 VI

	3.	Non-linear photophysical relevant data
	Cpd	2λmax 1PA [nm]	λmax1 2PA [nm]	σ2 [GM]	λmax2 2PA [nm]	σ2 [GM]
	QfB1	900	820	880	-	-
	QfvB1	1010	820	2972	890	2244
	QfeB1	948	820	2445	890	1884
	QftB1	1056	840	2783	890	2850

Table VI 5

 VI are gathered the relevant data concerning the FONs.

	Table VI 5.	Morphological and structural relevant data
	Cpd	dTEM [nm]	N [10 4 ]	ζ-potential [mV]
	QfB1	52	4.9	-67
	QfvB1	26	0.5	-64
	QfeB1	39	1.7	-74
	QftB1	57	4.9	-70
	QfeB2	19	0.2	-66

Table VI

 VI

	6.	Linear photophysical relevant data
	Cpd	λmax 1PA [nm]	εmax [10 4 M -1 cm -1 ]	λmax em [nm]	Φf	τ1 [ns]
	QfB1	458	2.7	600	0.32	2.4 (0.3) 5.4 (0.7)
						4.5 (0.1)
	QfvB1	505	9.5	650	0.09	1.6 (0.7)
						0.58 (0.2)
						4.6 (0.4)
	QfeB1	477	3.1	621	0.04	1.0 (0.3)
						17.5 (0.3)
	QftB1	534	4.9	690	0.08	1.1 (0.5) 2.4 (0.5)
	Comparing the quadrupoles in both solution and confined environment one can see, in Figure
	VI 9, an hyperchromic effect displayed upon nanoaggregation, except for QfeB1, indicating a
	cooperative effect between the neighbor chromophores within the nanoparticle. Moreover, one can
	observe a broadening of the ICT band that can be ascribed to an excitonic coupling after
	nanoconfinement. The excitonic coupling is more pronounced in FONs QfvB1, in fact one can see the
	formation of a shoulder suggesting an intermediate molecular arrangement between the two limit: J
	and H aggregates 29 .

  Table VI 7.

	Cpd	2λmax 1PA [nm]	λmax1 2PA [nm]	σ2 [GM]	λmax2 2PA [nm]	σ2 [GM]
	QfB1	916	840	1247	880	519
	QfvB1	1010	820	1764	890	1877
	QfeB1	954	820	1210	890	1650
	QftB1	1068	840	1945	890	2383

Table VI

 VI

	8.	Linear photophysical relevant data
	Cpd	λmax 1PA [nm]	εmax [10 4 M -1 cm -1 ]	λmax em [nm]	Φf	τ1 [ns]
						4.6 (0.4)
	QfeB1	477	3.1	621	0.04	1.0 (0.3)
						17.5 (0.3)
						4.8 (0.4)
	QfeB2	484	2.8	652	0.03	0.9 (0.3)
						17.8 (0.3)

Concerning the luminescent properties, increasing the number of electron acceptor moieties in the core of the quadrupole induces a bathochromic shift but, interestingly, the fluorescence quantum yield is not affected. On the other hand, comparing QfeB2 in both THF solution and FONs in water displayed in Figure

VI 12b, one

  can see that the nanoconfinement leads to an hypsochromic shift of the emission band but, remarkably, the FONs prepared do not display the quenching observed for all the other chromophores of the series.As reported in Table VI 8 both dyes in confined environment display three lifetimes in the same range, indicating that, in addition with the two "classical" lifetimes measured for the chromophores in the core and in the shell, these two dyes form upon aggregation a third emissive system that is probably related to a particular arrangement of molecules organized in a short range,

which display a long lifetime in the range of 17 ns (see Table

VI 8

).

Table VI

 VI

	9.	Non-linear photophysical relevant data
	Cpd	2λmax 1PA [nm]	λmax1 2PA [nm]	σ2 [GM]

  Table VI 10. Luminescence properties including one-photon and two-photon brightness of FONs in water

	Cpd	λmax 1PA [nm]	λmax em [nm]	FWHM em [10 3 cm -1 ]	Φf	εmaxΦf a) [10 8 M -1 cm -1 ]	λmax 2PA [nm]	σ2Φf a) [10 6 GM]
	QfB1	458	600	5.2	0.32	4.2	840	19.4
	QfvB1	505	650	4.4	0.09	0.4	890	0.9
	QfeB1	477	621	4.9	0.04	0.2	890	1.1
	QftB1	534	690	4.2	0.08	1.9	890	9.3
	QfeB2	484	652	5.3	0.03	0.01	900	0.07

  NMR (200 MHz, DMSO-d6): 8.03 (s, 1H,); 7.77 (d, J = 4Hz, 1H); 7.62-7.49 (m, 5H); 7.46 (d, J = 4Hz, 1H); 7.37-7.29 (m, 4H); 7.12-7.04 (m, 6H); 6.93 (d, J = 8Hz, 2H); 3.02 (br, 6H) ppm. CDCl3, 50 MHz) 164.0, 148.0, 147.3, 146.1, 145.7, 145.2, 137.6, 134.5, 134.1, 129.4, 127.2, 127.1, 126.6, 124.8, 123.6, 123.5, 123.3, 123.2, 116.8, 100.5, 38.1, 35.5 ppm.

1 H 13 C NMR (

  CDCl3, 50 MHz) 163.6, 148.2, 148.1, 147.2, 146.9, 146.4, 139.5, 133.8, 133.7, 129.4, 127.7, 126.9, 126.6, 124.9, 124.0, 123.6, 123.4, 123.0, 116.2, 96.7, 53.2 ppm.

	1 H NMR (200 MHz, DMSO-d6) 8.55 (s, 1H); 8.03 (d, J = 4Hz, 1H); 7.64-7.58 (m, 4H); 7.48 (d, J =
	4Hz, 1H); 7.37-7.29 (m, 4H); 7.09-7.04 (m, 6H); 6.97 (d, J = 8Hz, 2H); 3.82 (s, 3H) ppm.

13

C NMR (

  NMR (75 MHz, CDCl3) 147.84, 147.07, 146.49, 145.95, 134.18, 133.97, 132.58, 128.14, 127.69, 126.87, 126.00, 124.09, 123.80, 116.30, 116.18, 53.23.

	1 H NMR (200 MHz, DMSO-d6) 8.55 (s, 1H); 8.03 (d, J = 4Hz, 1H); 7.68-7.59 (m, 4H); 7.54-7.46
	(m, 5H); 7.05-6.98 (m, 6H); 3.82 (s, 3H) ppm.

13 C

  CDCl3, 50 MHz) 150.1, 149.5, 147.3, 147.2, 145.9, 140.3, 133.6, 133.2, 132.6, 128.5, 127.8, 126.9, 126.1, 124.2, 123.9, 123.6, 116.4, 114.4, 113.5, 75.7 ppm. an Erlenmeyer flask and was stirred for 1h neutralizing the I2 in excess. The mixture was filtered and the solid washed with dichloromethane and dried at 85°C overnight. A brown solid was obtained (yield = 63%).

	Melting Point = 152°C;
	1 H NMR (200 MHz, DMSO-d6) 8.61 (s, 1H); 7.89 (d, J = 4Hz, 1H); 7.70-7.63 (m, 4H); 7.55-7.46
	(m, 5H); 7.04-6.98 (m, 6H) ppm.

13

C NMR (FTIR (ATR): ν (CHarom/HC=C) 3021 cm -1 ; ν (C≡N) 2210 cm -1 . in

13 C NMR

  (75 MHz, CDCl3): 182.71, 155.21, 151.88, 147.57, 147.45, 147.36, 143.61, 143.17, 142.38, 141.99, 139.57, 137.54, 133.98, 131.72, 129.34, 129.27, 128.29, 126.43, 126.38, 125.62, 124.69, 124.57, 124.07, 123.81, 123.62, 123.20, 123.17, 123.02, 120.57, 120.54, 120.31, 119.63, 77.23, 55.15, 38.20, 32.55, 28.21, 22.39, 22.36.

	HRMS (m/z) [M]calcd for C50H47N1O1S2: 741.30991, found: 741.31082

A special acknowledgement is going to Nano2Fun (European Union's Seventh Framework Programme FP7/n°607721) that gave me the opportunity to do this challenging experience. Thanks to the Nano2Funners, friendly people and bright scientists with whom I shared the project, travelling from the resort in Slupsk

most allowed band is markedly blue-shifted compared with twice the wavelength of the ICT band of the linear process, paralleling the behavior observed in CHCl3 solution.

Furthermore, it can be observed in Figure III 11d that within FONs the rigidification of the πconjugated bridge as well as the addition of a hindered fluorene core in the connector, induce an improving of the 2P response.

Molecular confinement

In this paragraph will be discussed the differences of the optical properties of chromophores in both molecular solution and organic nanoparticles. So far, intensive studies were done to understand the behavior of one-photon processes of molecules upon nano-confinement inducing either aggregation caused quenching (ACQ) or aggregation induced enhanced emission (AIEE) 12,13 , however the "world" of NLO is still to be fully discovered. As discussed previously (cf. Paragraph 1:

Introduction), the arrangement of molecules within nanoparticles can strongly affect the photophysical properties.

Here I would like to display a complete overview of the behaviors of dipolar chromophores before and after the nanoaggregation process. Due to the big amount of dipolar dyes, I chose few emblematic samples to give an exhaustive review of the observations done.

Linear optical properties

Emission in solid state and upon nanoaggregation

As I wrote earlier in the chapter (cf. Paragraph 2), the nanoparticles are formed under a kinetic domain and I guess that it is worth to compare the photophysical behavior of the molecules in solid state (smeared powders, microcrystalline when it was possible) and in nanoparticle. It is interesting to highlight the fact that these chromophores, even in solid state, maintain their luminescent properties without undergoing any quenching of fluorescence due to close intermolecular interactions.

CHAPTER 4 CHARGED DIPOLES 104

To decrease the probability of close interactions between neighbor dipoles, the idea was to synthesize multi-dipolar chromophores in which the active subunits are connected by a rigid πconjugated bridge, as displayed in Illustration V 1. The propeller shape of the diphenylamine associated with the rotational motion of the different part of the molecules give us the opportunity to study these chromophores upon nano-confinement hoping an AIE/AIEE phenomenon.

The short dipoles were prepared using a substituted phenylamine as donor moiety and either aldehyde (CHO) or dicyanovinyl (DCV) as electron-withdrawing end-groups. The dipolar subunits were linked together by a fluorene or a biphenyl core in a quadrupolar-like arrangement, or with a truxene core in a octupolar-like configuration. It was used as monomer a push-pull dipole linked simply with a fluorenyl moiety. In

Structural characterization of the articulated dipoles

The chromophores studied in this chapter were synthesized by Prof. Jean-Baptiste Verlhac (JBV), thus the chemical synthesis will not be explained in detail here. In Scheme V 1 you can find the general synthetic route followed.

Synthetic route of the chromophores

Crystal characterization

In this paragraph are described the crystal structure obtained for the chromophores 1Bis(F), 2Bis(F) and 1Tris. As will be discussed, these molecules in the crystal packing did not display specific interactions and, thanks to the presence of hindering groups, the close π-π stacking is prevented. Thus, one can expect weak intermolecular interaction that will be an advantage for photophysical properties, when the molecule is confined in nanoparticle.

Articulated dipoles: an essential state model description

The essential state model is an interesting tool that allows to simplify the description of electronic states, choosing the main resonance structures as basis states, for the investigation of linear and non-linear optical properties in molecules that are formed from electron-releasing and electronwithdrawing end-groups connected by a π-conjugated bridge.

When dealing with dipolar dyes, the easiest description is to take into account two basis wavefunctions: the neutral and the zwitterionic resonance structures [19][20][21][22] .

Representative illustration of the two basis states of a dipolar dye:

the neutral |𝑁〉 and the zwitterionic |𝑍〉 Both ground |𝐺〉 and excited |𝐸〉 state of chromophore will be linear combinations of these two basis states, depending on the strength of both the electron-withdrawing and electron-releasing moieties and on the specific structure, particularly the ability of the π-conjugated bridge to make the charge resonate. The energy gap between the two basis states is 2 𝑧 0 while the mixing element is

Thanks to the large brightness upon both one and two-photon absorption, and the emission in the biological transparency window of these nanoparticles it was possible to use them in bioenvironment as fluorescent probes, where they showed low cytotoxic activity. Commercially available reagent (purchased from Aldrich, Alfa Aesar and TCI) were used without further purification. Dry solvents were distilled from the appropriate drying reagents immediately before use. Compounds already described in the literature were characterized by 1 H NMR spectra and melting points and compared to reported data. All air-or water-sensitive reactions were carried out under argon. Column chromatography was performed using Fluka silica gel Si 60 (40-63 µm, 230-400 mesh).

Chemical characterization

Melting points were determined in a STUART SMP 10 digital melting point instrument.

Infrared spectra were measured on a Perkin Elmer Spectrum 100 Optica. 1 H and 13 C NMR spectra were recorded on a Bruker Advance III 200 (400) spectrometer at 200 (400) MHz and 50 (100) MHz respectively. Mass spectra were performed by the CESAMO (Bordeaux, France). X-ray diffraction measurements on single crystals D1 ' b, D1 ' c and D2 ' b were performed on a Bruker APEX II diffractometer at CESAMO platform (Bordeaux, France), while measurement on single crystals 1D ''' a, 1Bis(F), 2Bis(F), 1Tris, QfvB1 and QfeB1 were performed on a FR-X Rigaku diffractometer at IECB (Bordeaux, France). The structures were solved with SHELXS97 and refined by a full-matrix leastsquares method on F 2 using SHELXL-97 1 . All non-hydrogen atoms were refined with anisotropic displacement parameters. Hydrogen atoms were placed at calculated positions using suitable riding models. Their positions were constrained relative to their parent atom using the appropriate HFIX command in SHELXL-97. Additional disordered solvent molecules were found in the lattice of 1D ' c (THF) and QfvB1 (DCM), unfortunately they could not be refined and thus the SQUEEZE command was applied.

To span the 1000-1200 nm range, an OPO (PP-BBO) was added to the setup to collect and modulate the output signal of the Ti:Sapphire oscillator. The excitation was focused into the cuvette through a microscope objective (10X, NA 0.25). The fluorescence was detected in epifluorescence mode via a dichroic mirror (Chroma 675dcxru) and a barrier filter (Chroma e650sp-2p) by a compact CCD spectrometer module BWTek BTC112E. Total fluorescence intensities were obtained by integrating the corrected emission. The experimental uncertainty of the absorption cross-section values determined from this method has been estimated to be ±10%.

Cellular imaging and cytotoxicity

For the fluorescence imaging experiments, COS 7 cells were cultured on glass disks, using DMEM supplemented with 10% Fetal Calf Serum, as culture medium. When the cells reached 30% confluence, the FONs were added to the culture medium at a final concentration of dye of 1 µM (equivalent to a nM concentration of nanoparticles). After 8h of incubation, the cells were abundantly washed with PBS, fixed with 4% para-formaldehyde and mounted using a Fluoromount TM media (Sigma). The fluorescence imaging was performed at Bordeaux Imaging Center using a Leica DM6000 confocal microscope. For the co-localization experiments, the cells were incubated simultaneously with both FONs, at equimolar concentration (final concentration in chromophore of 1µM).

For the toxicity test, cells were pleated in 96 wells culture plate and grown in DMEM culture medium until approximately 80% confluence. At this point, the culture medium was replaced by a new one, enriched with FONs at a final concentration of dye of 1µM. The cytotoxicity was determined after 24h of incubation with the FONs by MTT assay based on the conversion of tetrazolium salt (MTT) into a purple formazan product, using the iMark TM microplate reader (BioRAD).

2-[ [5'-[4-(diphenylamino)phenyl] [2,2'-bithiophen] -5-yl] methylene] malononitrile (D1 ' d):

Following the general procedure using D1 ' a as aldehyde and malononitrile as α-acid derivatives to yield compound D1 ' d as a dark red solid (yield = 39%). Monocrystals suitable for X-ray diffraction were obtained by slow evaporation of a CH2Cl2 solution.

Melting Point: 183°C

Melting Point: 148°C. the first flask were added under Ar in the second flask and the reaction was left in reflux for 48h. The reaction was quenched with ice and extract with diethyl ether. The organic phase was dried under reduced pressure. The crude was purified by a recrystallization in cyclohexane. A white solid was isolated (yield = 88%).

Melting Point = 138-139°C. 5): 8 mL of dichloroethane were added under Ar to 4 (1.48 mmol, 1 eq.), POCl3 (2.97 mmol, 2 eq.) and distilled DMF (12 mmol, 8 eq.) at 0°C. The reaction mixture was warmed to RT and stirred for 4h, then it was heated up to reflux for 20h. The reaction was quenched with the addition of 45 mL of a saturated solution of sodium acetate. The crude was extracted with dichloromethane, and then concentrated under reduced pressure. The crude was purified by recrystallization in iPrOH. A yellow-green crystals were isolated (yield = 42%).

Melting Point = 161-162 °C. 72, 155.20, 151.91, 151.88, 144.95, 142.36, 142.04, 139.70, 137.53, 134.00, 131.79, 128.16, 125.63, 125.17, 124.83, 123.84, 123.14, 120.60, 120.54, 120.36, 120.16, 55.18, 38.19, 32.57, 28.21, 22.38 ppm.

5-(7-(5-iodothiophene-2-yl)-9,9-diisopentyl 9H fluorene-2-yl)thiophene-2-carbaldehyde (6):

In a round bottom flask were added 200 μL of water, 300 μL of acetic acid, 6.6 μL of sulfuric acid, 5 (0.206 mmol, 1 eq.) and periodic acid (1.44 mmol, 7 eq.) under stirring. In the meanwhile a solution of iodine (0.206 mmol, 1 eq.) in 1.1 mL of carbon tetrachloride was prepared and added dropwise to the reaction mixture. The temperature was increased to 80°C for 1h, then was cooled to RT and the reaction was kept under stirring overnight. The reaction mixture was neutralized with a saturated solution of NaHCO3 and extracted with dichloromethane. The organic phase was washed with a saturated solution of Na2S2O3 and dried under reduced pressure. Yellow-green crystals were obtained after recrystallization from iPrOH (yield = 74%).

Melting Point = 168-169 °C.