Jean Rouillon

† Lamiaa

M A Ali

Kamel Hadj-Kaddour

Raphaël Marie-Luce

Guillaume Simon

Mélanie Onofre

Sandrine Denis-Quanquin

Marion Jean

Muriel Albalat

Nicolas Vanthuyne

Guillaume Micouin

Akos Banyasz

Magali Gary-Bobo
email: magali.gary-bobo@inserm.fr

Cyrille Monnereau
email: cyrille.monnereau@ens-lyon.fr

Chantal Andraud

Assembly of Aggregation-Induced Emission Active Bola-Amphiphilic Macromolecules into Luminescent Nanoparticles Optimized for Two-Photon Microscopy In Vivo

The (Z) and (E) isomers of an extended tetraphenylethylene-based chromophore with optimized two-photon induced luminescence properties are separated and functionalized with watersolubilizing pendant polymer groups, promoting their self-assembly in physiological media in the form of small, colloidal stable organic nanoparticles. The two resulting fluorescent suspensions are then evaluated as potential two-photon luminescent contrast agents for intravital epifluorescence and two-photon fluorescence microscopy. Comparisons with previously reported

works involving similar fluorophores devoid of polymer side chains illustrate the benefits of the later functionalization regarding the control of the self-assembly of the nano-objects, and ultimately, their biocompatibility towards the imaged organism.

INTRODUCTION

Since its invention in the late 1990's two-photon fluorescence microscopy has experienced impressive developments, [START_REF] Helmchen | Deep tissue two-photon microscopy[END_REF][START_REF] So | Two-Photon Excitation Fluorescence Microscopy[END_REF] and has opened unprecedented perspectives in tissues imaging that have substantially benefited such diverse fields as neurobiology, [START_REF] Ellis-Davies | Two-Photon Microscopy for Chemical Neuroscience[END_REF] physiology, [START_REF] Shih | Two-Photon Microscopy as a Tool to Study Blood Flow and Neurovascular Coupling in the Rodent Brain[END_REF] embryology 5 and tissue engineering. [START_REF] Schenke-Layland | Two-photon microscopes and in vivo multiphoton tomographs -Powerful diagnostic tools for tissue engineering and drug delivery[END_REF] These developments have been achieved thanks to combined advances in the optical setups design and in the developments of increasingly sophisticated and selective endogenous (green fluorescent proteins) [START_REF] Ibraheem | Designs and applications of fluorescent protein-based biosensors[END_REF] and exogenous (synthetic organic chromophores and nanoparticles) fluorescent contrast agents. [START_REF] Miller | Deep tissue imaging with multiphoton fluorescence microscopy[END_REF][START_REF] Drobizhev | Two-photon absorption properties of fluorescent proteins[END_REF][START_REF] Yao | Two-Photon Fluorescent Probes for Bioimaging[END_REF][START_REF] Lou | Organic Dots Based on AIEgens for Two-Photon Fluorescence Bioimaging[END_REF] In this framework, the conception of molecular probes or nano-objects that combine sizeable two-photon induced emission properties with good solubility (or colloidal stability) in physiological media has been a major issue, on which we have been extensively working in recent years. [START_REF] Adjili | Biocompatible photoresistant far-red emitting, fluorescent polymer probes, with near-infrared two-photon absorption, for living cell and zebrafish embryo imaging[END_REF][START_REF] Bui | Near infrared two photon imaging using a bright cationic Yb(iii) bioprobe spontaneously internalized into live cells[END_REF][START_REF] Navarro | Nanocarriers with ultrahigh chromophore loading for fluorescence bio-imaging and photodynamic therapy[END_REF] In previous reports, we have proposed chromophore functionalization with water solubilizing poly(hydroxyethylacrylate) (PHEA) polymeric side chains as a relevant approach to achieve high biocompatibility of molecular bioprobes. [START_REF] Monnereau | Water-soluble chromophores with star-shaped oligomeric arms: synthesis, spectroscopic studies and first results in bio-imaging and cell death induction[END_REF] We have shown that this functionalization could efficiently shield the molecule from interactions with its local environment, ensuring solubility of inherently lipophilic and prone-to-stacking fluorophores into aqueous media, and keeping luminescence efficiencies high. This strategy has been used in the making of fluorophores that have already found promising application for diverse bio-related applications such as intravital imaging of cerebral vascularization [START_REF] Massin | A water soluble probe with near infrared two-photon absorption and polarity-induced fluorescence for cerebral vascular imaging[END_REF][START_REF] Pascal | Keto-polymethines: a versatile class of dyes with outstanding spectroscopic properties for in cellulo and in vivo two-photon microscopy imaging[END_REF][START_REF] Mettra | A Fluorescent Polymer Probe with High Selectivity toward Vascular Endothelial Cells for and beyond Noninvasive Two-Photon Intravital Imaging of Brain Vasculature[END_REF] or the detection of biomolecules (ds-DNA, [START_REF] Deiana | Interactions of a biocompatible water-soluble anthracenyl polymer derivative with double-stranded DNA[END_REF] Gquadruplexes [START_REF] Deiana | Specific Recognition of G-Quadruplexes Over Duplex-DNA by a Macromolecular NIR Two-Photon Fluorescent Probe[END_REF][START_REF] Deiana | Unravelling the Binding Mechanism of a Poly(cationic) Anthracenyl Fluorescent Probe with High Affinity toward Double-Stranded DNA[END_REF] and proteins [START_REF] Deiana | Two-Photon Macromolecular Probe Based on a Quadrupolar Anthracenyl Scaffold for Sensitive Recognition of Serum Proteins under Simulated Physiological Conditions[END_REF] ) in complex physiological media. In parallel, we have recently reported on extended tetraphenylethylene (TPE) derivatives -a class of molecules classically used for their Aggregation-Induced Emission (AIE) properties [START_REF] Yang | Recent advances in mechano-responsive luminescence of tetraphenylethylene derivatives with aggregation-induced emission properties[END_REF][START_REF] Leung | Restriction of Intramolecular Motions: The General Mechanism behind Aggregation-Induced Emission[END_REF][START_REF] Zhao | Aggregation-Induced Emission: New Vistas at the Aggregate Level[END_REF] in particular for bioimaging and biosensing applications [START_REF] Lin | Novel near-infrared II aggregation-induced emission dots for in vivo bioimaging[END_REF][START_REF] Tong | Fluorescent "light-up" bioprobes based on tetraphenylethylene derivatives with aggregationinduced emission characteristics[END_REF][START_REF] Zhu | Aggregation-Induced Emission Materials with Narrowed Emission Band by Light-Harvesting Strategy: Fluorescence and Chemiluminescence Imaging[END_REF][START_REF] Wang | Temperature-Sensitive Fluorescent Organic Nanoparticles with Aggregation-Induced Emission for Long-Term Cellular Tracing[END_REF][START_REF] Li | A novel cross-linked nanoparticle with aggregation-induced emission properties for cancer cell imaging[END_REF][START_REF] Zhang | Fluorescent Polymersomes with Aggregation-Induced Emission[END_REF] -that had been specifically designed to maximize the two-photon emission properties of their aggregates. [START_REF] Rouillon | Two-Photon Absorbing AIEgens: Influence of Stereoconfiguration on Their Crystallinity and Spectroscopic Properties and Applications in Bioimaging[END_REF] In the course of this study, it turned out that, in spite of the excellent colloidal stability and excellent spectroscopic properties of the suspension obtained by nano-precipitation of TT OMe , only limited control over the assembly was achieved : as a result, mixtures of large (d>100 nm) and disperse nanoparticles were obtained with detrimental outcome on the long-term survival of the imaged zebrafish. It has already been established in previous literature reports that the self-assembly of fluorophores containing macromolecules (either in the form of grafted polymers or conjugated polymers) usually led to smaller and better controlled self-assembly compared to dye-loaded nanoobjects obtained by conventional nano-precipitation methods. [START_REF] Reisch | Fluorescent Polymer Nanoparticles Based on Dyes: Seeking Brighter Tools for Bioimaging[END_REF] In particular, direct self-assembly of fluorescent amphiphilic polymers upon dispersion in aqueous media has proved to be a remarkably effective approach for the synthesis of small (10-14 nm) monodisperse nanoparticles, well-suited for in vivo applications, [START_REF] Collot | Stealth and Bright Monomolecular Fluorescent Organic Nanoparticles Based on Folded Amphiphilic Polymer[END_REF] while larger nanoparticles (> 100 nm, typically) are rapidly cleared from the bloodstream upon internalization within macrophages and tend to induce more severe adverse effects such as acute inflammations. [START_REF] Gamucci | Biomedical Nanoparticles: Overview of Their Surface Immune-Compatibility[END_REF] In this work, wishing to further explore extended TPE derivatives and taking advantage of our above-mentioned long-time expertise in the functionalization of lipophilic fluorophores through controlled radical polymerization, we have synthesized and studied TT P a new two-photon absorbing, water-soluble and AIE active luminescent probe, that can be derived into two stereoisomers differing by the stereoconfiguration of their central double bond (Fig. 1). Through a combined study, involving DLS and DOSY NMR, we investigate the self-assembly process of both isomers and their mixture in aqueous suspension. In particular, we show that, owing to the amphiphilic macromolecular nature of the molecule, it is possible to obtain controlled small-sized (ca 10 nm) nano-assemblies of the chromophores under either their (Z) and (E) forms in both pure water and physiological media. The resulting colloidal suspensions combine significant twophoton absorption (2PA) cross sections and Aggregation-Induced Emission efficiencies, making them suitable for two-photon fluorescence microscopy in living biological systems. Comparison with our previously studied assemblies of pure nano-precipitated (E/Z)TT OMe chromophores 32 on several days after injection into zebrafish embryos clearly demonstrated the benefits of polymer functionalization and underlined the biocompatibility of these resulting nano-objects.

MATERIALS AND METHODS

Synthesis

Syntheses were performed in oven-dried vessel purged with argon. Solvents were dried on 3 Å molecular sieves. The reactions were monitored by analytical thin layer chromatography (TLC) on Merck 60 F-254 precoated silica gel plate (0.2 mm thickness) and product were revealed at UV lamp. Purification by column chromatography was made using over Mercksilica gel 60 (0.040-0.063 mm).

All newly synthesized compounds were routinely characterized by 1 H and [START_REF] Bui | Near infrared two photon imaging using a bright cationic Yb(iii) bioprobe spontaneously internalized into live cells[END_REF] C NMR, and highresolution mass spectrometry (HRMS). Detailed synthetic protocols and characterization data, including NMR spectra, are available as supplementary material (S1 and S2, Fig. S2-S15)

NMR studies

NMR spectra were recorded at room temperature on a Bruker Avance 400 functioning at 400 MHz. Chemical shifts are reported as values (ppm) with reference to the peak of CDCl3, DMSO-d6, or D2O. Abbreviations for the 1 H NMR data are: chemical shift δ, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet), coupling constants J.

Diffusion experiments were performed on a Bruker Avance III 400 MHz spectrometer equipped with a Prodigy Cryoprobe. The temperature was regulated at 298K. A bipolar pulse longitudinal eddy current delay (BPPLED) pulse sequence was chosen, and the diffusion time was set to 250 ms. Sine shaped pulse field gradient were used with a length of 2.5 ms and the gradient intensity was linearly incremented from 0.963 to 47.187 G cm-1 over 30 experiments. After each gradient pulse, a 200 ms delay was used as a recovery delay. DOSY NMR were generally processed by Mestrelab MNova using bayesian transformation.

Polydispersity of the polymer was determined thanks to the maximum entropy methodology, processed on NMRNotebook. [START_REF] Viéville | Polydispersity index of polymers revealed by DOSY NMR[END_REF] Dynamic light scattering (DLS) was recorded with a Malvern Instruments Zetasizer nano series instrument (with solutions at constant 10 -5 M concentration).

HRMS measurements were performed at the Centre Commun de Spectrométrie de Masse (CCSM) of Lyon (France).

Preparative separation of the (E) and (Z) isomers

The separation of the (E) and (Z) isomers was done on the compound (E/Z)TT In using a chiral stationary phase, an amylose tris-(3,5-dimethyl-phenylcarbamate). Sixty successive injections on Chiralpak IA (250 x 10 mm), with hexane/2-PrOH/dichloromethane (90/5/5) as mobile phase at 5 mL.min -1 , allowed to obtain 29 mg of the first eluted isomer, (Z)TT In , and 38 mg of the second eluted isomer, (E)TT In (Chromatograms on Fig. S16).

Surface Tension (SFT) measurements

Six aqueous solutions of (E/Z) TTP were prepared from successive dilutions of a 42.7 μmol/L solution of the polymer surfactant in ultrapure water (HPLC grade, Sigma-Aldrich). Prior to the SFT measurements, their density was measured with a DMA 5000M densimeter from Anton Paar.

The surface tension of the 7 polymer surfactant solutions was measured by the pendant drop method, with a DSA 30E tensiometer, from Krüss, controlled by the supplier's software, Advance.

The pendant drop method is an optical way to obtain the SFT of a liquid in a gas phase, by solving the Young-Laplace equation: 37

∆𝑃 = ∆𝑃 0 -∆𝜌𝑔𝑧 = 𝜎 ( 1 𝑅 1 + 1 𝑅 2 )
with σ the surface tension, R1 and R2 the radii of the curvature, g the gravitational constant, z the spatial coordinate, Δρ the density difference between the liquid phase and the gas phase, ΔP the pressure difference, and ΔP0 the reference pressure at z = 0. The Young-Laplace equation can be expressed in the cylindrical referential with 3 equations giving the coordinates, by the use of the drop shape factor B:

𝐵 = ∆𝜌𝑔𝑅 0 2 𝜎
with R0 the radius of the curvature at the apex of the drop. The computational Young-Laplace fit of the drop shape allows to calculate B, and thus the surface tension σ.

For the SFT measurements of each concentration, a 500 µL glass Hamilton syringe was filled with the solution and vertically positioned in the tensiometer. The syringe plunger was slowly pushed by an automated program, in order to form progressively a drop at the tip of a stainless-steel needle (ø = 1.826 mm). From the image taken with the camera of the system, the calibration made with the diameter of the needle, and the known densities of the air phase and the liquid, the software could analyze the drop shape and solve the Young-Laplace equation. The surface tension was measured at regular time intervals until the drop went out of the frame. Two or three drops were analyzed for each solution and the stable data points were kept, so that it was possible to obtain a consistent average of the surface tension, from a high number of data points (> 100 points).

Spectroscopic measurements

Absorption spectra (UV-Vis) were recorded with a Jasco 670 spectrometer. Fluorescence spectra were performed on a Horiba Jobin-Yvon Fluorolog-3 spectrofluorometer. Spectra were referencecorrected for both the excitation source light intensity variation (lamp and grating) and the emission spectral response (detector and grating). Emission spectra were recorded at 350 nm as excitation wavelength. For the comparison of emission spectra of compound in various water/DMSO mixtures, spectra were normalized of their respective absorbance. Absolute fluorescence quantum yields were measured with a calibrated integrative sphere (2π steradians covered with spectralon®, model G8 from GMP) as described in the literature [START_REF] Helmchen | Deep tissue two-photon microscopy[END_REF] . Each sample was placed on a quartz tube at the concentration of 2.10 -5 M. Four measurements were made with constant excitation and emission to give four integrated intensities. E c and E a are integrated fluorescence spectra of direct excitation with and without the sample respectively. L c and L a are integrated excitation spectra of the sphere with and without the sample respectively. A density filter (Transmittance = 0.5 %) was used to reduce the intensity of excitation profile. Quantum yield is calculated according to the equation ( 1):

Φ fluo = E c -E a f (L a -L c ) (1)

Two-photon absorption cross sections measurements

Two-photon excitation spectra of the studied compounds were measured by two-photon excitation-induced fluorescence technique in the 700-900 nm range. Excitation source was a tunable femtosecond Ti:sapphire laser (Chameleon Ultra I, Coherent). Laser beam was focused into a 10x10 cm quartz (QZS) cell using a 100 mm focal length lens. The cell containing the sample was positioned to minimize the optical path of the exciting laser and that of the emitted fluorescence in the sample thus minimizing inner filter effects and other artefacts. Emitted fluorescence was collected at 90° with respect to the excitation, filtered by adapted low-pass filters and was detected by a fiber optic spectrometer (AvaSpec-EVO, Avantes).

Cell culture

Human breast cancer cells MCF-7 (purchased from ATCC) were cultured in DMEM/F-12 medium with GlutaMAX™-I (containing 4.5 g.L -1 of D-glucose) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Cells were grown at 37 °C, in humidified atmosphere with 5% CO2.

Cytotoxicity study

MCF-7 cells were seeded in 96-well plate, incubated with increasing concentrations (from 2.5 to 150 µg.mL -1 ) of the different isomers for 3 days and the percentage of living cells was quantified by MTT assay. This consists in a colorimetric test performed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Promega) where cells were incubated with 0.5 mg•mL -1 MTT for 4 h to determine mitochondrial enzyme activity. Then, MTT precipitates were dissolved in 150 µL ethanol/DMSO solution (in proportion 1/1) followed by 20 min of shaking and absorbance was read at 540 nm. Cells treated with the vehicle were served as control.

Imaging potential in vitro

The cellular uptake experiment was performed using confocal fluorescence microscopy on living cells. Cells were seeded (10 6 cells•cm -2 ) on culture dishes with a glass bottom (Fluorodish from World Precision Instruments, Stevenage, UK). After seeding, cells were incubated with 50 µg.mL -1 of (E) or (Z) isomers for 24 h. Cells were also loaded 15 min with CellMask TM (Invitrogen, Cergy Pontoise, France) at 5 µg•mL -1 , for membrane staining. Then, cells were washed twice with culture medium and fluorescence imaging was performed on living cells with a LSM780 LIVE confocal microscope (Carl Zeiss, Le Pecq, France), at 750 nm for isomers detection and 561 nm for cell membranes. All images were obtained with a high magnification (Plan-Apochromat 40x/1.3 Oil DIC).

Zebrafish breeding

Casper zebrafish embryos were purchased from Zebrafish International Resource Center (ZIRC)

and raised to adulthood in circulating aquarium system at 28 °C and 14 h light/10 h dark cycle, in the Molecular Mechanisms in Neurodegenerative Dementia (MMDN) laboratory (Inserm U1198), Montpellier University. Experiments with zebrafish embryos are performed according to the EU Directive 2010/63/EU on the protection of animals used for scientific purposes.

Imaging on Casper zebrafish embryos

Casper zebrafish embryos of 72 h post fertilization (hpf) were intravenously injected with 10 nL of (Z) or (E) isomer at 1 mg.mL -1 concentration (n = 7 for control, n = 9 for (Z) isomer, n = 10 for (E) isomer). One hour after injection, embryos were observed using one-photon excitation microscope at excitation wavelength of 340-380 nm, and after embryos were observed using twophoton excitation confocal microscopy at excitation wavelength of 750 nm.

Toxicity study on zebrafish embryos

After injections, embryos were then maintained 3 days in breeding conditions to analyze every day the biocompatibility of (Z) isomer and (E) isomer according to various parameters relating the good development of embryos, such as mobility, heart rate, malformations and deaths.

RESULTS AND DISCUSSIONS

Synthesis

All molecules discussed in the following are presented in Figure 1. Synthesis of the (E/Z)TT In macroinitiator was adapted from the method previously published by us for the (E/Z)TT OMe molecule, [START_REF] Rouillon | Two-Photon Absorbing AIEgens: Influence of Stereoconfiguration on Their Crystallinity and Spectroscopic Properties and Applications in Bioimaging[END_REF] keeping here the phenolic function free from the beginning (Scheme S1, detailed synthetic procedures available as supplementary materials, S1). Thus, Buchwald-Hartwig coupling between 4-hydroxydiphenylamine and para-bromo-iodobenzene was achieved with a good yield to afford 4-bromo-N-(4-hydroxyphenyl)diphenylamine (1), thanks to a procedure that made it compatible with the presence of the free phenolic moieties. [START_REF] Harris | Improved Functional Group Compatibility in the Palladium-Catalyzed Synthesis of Aryl Amines[END_REF] Then, a palladium catalyzed Sonogashira cross-coupling with (triisopropylsilyl)acetylene yielded alkyne 2, which was in turn deprotected with TBAF. The instable free alkyne as obtained was directly engaged in Sonogashira coupling with TPE-Br (Scheme S1) to lead to (E/Z)TT OH .

Finally, esterification between the (E/Z)TT OH diol and 2-bromo-2-methylpropionyl leads to the target macroinitiator (E/Z)TT In . This synthesis being non-stereoselective, separation of the macroinitiator diastereoisomers was required to afford stereopure versions of the chromophore. It was herein performed on amylose-based chiral stationary phase (Fig. S16), which efficiency has already been demonstrated by us on similar chromophores. [START_REF] Rouillon | Two-Photon Absorbing AIEgens: Influence of Stereoconfiguration on Their Crystallinity and Spectroscopic Properties and Applications in Bioimaging[END_REF] The controlled polymerizations of (E/Z)TT In as a (E/Z)-mixture, and also in stereopure form, were carried out in bulk 2-hydroxyethyl acrylate (HEA) monomer, using 2,2'-bipyridine and copper(I) bromide as a catalytic system, as already described in previous reports for the elaboration of related polymer-chromophore adducts. [START_REF] Monnereau | Water-soluble chromophores with star-shaped oligomeric arms: synthesis, spectroscopic studies and first results in bio-imaging and cell death induction[END_REF][START_REF] Massin | A water soluble probe with near infrared two-photon absorption and polarity-induced fluorescence for cerebral vascular imaging[END_REF][START_REF] Pascal | Keto-polymethines: a versatile class of dyes with outstanding spectroscopic properties for in cellulo and in vivo two-photon microscopy imaging[END_REF][START_REF] Mettra | A Fluorescent Polymer Probe with High Selectivity toward Vascular Endothelial Cells for and beyond Noninvasive Two-Photon Intravital Imaging of Brain Vasculature[END_REF] The characteristic signatures of the aromatic part of the 1 H NMR spectra confirm that stereopurity has been preserved after the ATRP reaction and purification (Fig. S13). Measurements of chain lengths and evaluation of of polydispersity were achieved by [START_REF] Helmchen | Deep tissue two-photon microscopy[END_REF] H NMR(Fig. S14) and 1 H DOSY NMR (Fig. S18), respectively, which will be discussed in the corresponding NMR study section. In all polymerization cases reported herein, a rigorous control of the polymerization kinetics makes it possible to obtain a range of target chain lengths in a controlled manner with low polydispersity (vide infra) which is essential for the comparison of the systems.

Spectroscopic studies

Then, the spectral features of (E/Z)TT P (n=16) were studied in DMSO. The absorption and emission spectra are presented in Figure 2, while main spectroscopic data are gathered in Table 1.

First, we notice that the spectral signatures of (E/Z)TT P (Fig. 2) correspond well to those observed for their parent monomeric equivalent (E/Z)TT OMe which can be summarized as follows: (i) a structureless ICT band, typical of large π-conjugated system; (ii) large Stokes shift (ca 6500 cm -1 )

indicating large reorganization processes between ground and excited electronic states; (iii) an absorption band of the (Z)-isomer slightly blue-shifted (λabs = 357 nm) with respect to (E)-isomer (λabs = 374 nm), and a reversed behavior for the emission with λem = 543 nm for the (Z)-isomer, 530 nm for the (E)-isomer of TT P ; (iv) a similar two-photon absorption cross section σ2PA for the (Z)-isomer as for the (E)-isomer (140 vs. 100 GM) in the same range of wavelengths maxima (ca 710 nm). Consequently, functionalization of this chromophore with the polymeric PHEA chains does not markedly affect its main spectroscopic characteristics in DMSO solutions. The fluorescence quantum yields Φf of TT P (stereopure Z and E form, and (E/Z) mixture) were also first estimated in DMSO solution, with the aim to highlight the influence of the polymerization degree on this parameter. Indeed, while the quantum yield of the macroinitiator (n = 0 in Fig. S17)

is ca Φf = 0.004, the addition of polymer chains leads to a moderate yet significant improvement to reach Φf = 0.07, a value that subsequently remained constant, within experimental error, for polymer chain lengths between 7 and 70 monomer units (Fig. S17). Thus, while the introduction of polymer chains seems to favor the emission (probably owing to steric-induced restriction of the rotation around the central double, a well-known mechanism in viscosity probes [START_REF] Ma | A minireview of viscosity-sensitive fluorescent probes: design and biological applications[END_REF] ) the effect quickly saturates as the polymer chain is extended, and emission quantum yield subsequently becomes independent on the length of the PHEA chains. In the rest of this study, we chose to focus on (E/Z)TT P with an average polymer chain length n = 16 (as calculated through comparison of the integration of terminal and main-chain signals, Fig S14 ), which offers good dispersibility in aqueous media with a limited impact on the molecular weight of the chromophore

The spectroscopic properties of the PHEA polymers in aqueous media were then studied in DMSO/water mixtures with increasing water volumic fraction (Fig. 3). For a volumic fraction in water fH2O lower than 50%, emission properties of (E/Z)TT P remain virtually constant with Φf ≈0.07. For fH2O above 50%, a hypsochromic shift from 520 to 467 nm is noticed, consistent with that observed for the parent monomeric fluorophore in similar conditions; this is accompanied by a marked and progressive increase in Φf with further addition of water, to reach a maximal Φf = 0.35 for a fH2O of 80% (Figure 3, Figure S. These two properties (hypsochromic shift and Φf increase) are compatible with the hypothesis of a restriction of the rotation phenomena around the central double bond [START_REF] Rouillon | Reevaluating the Solution Photophysics of Tetraphenylethylene at the Origin of their Aggregation-Induced Emission Properties[END_REF] resulting from interchromophore aggregation in aqueous media. Thus, in contrast with previously reported similar chromophore polymer adducts, [START_REF] Monnereau | Water-soluble chromophores with star-shaped oligomeric arms: synthesis, spectroscopic studies and first results in bio-imaging and cell death induction[END_REF][START_REF] Massin | A water soluble probe with near infrared two-photon absorption and polarity-induced fluorescence for cerebral vascular imaging[END_REF][START_REF] Pascal | Keto-polymethines: a versatile class of dyes with outstanding spectroscopic properties for in cellulo and in vivo two-photon microscopy imaging[END_REF][START_REF] Mettra | A Fluorescent Polymer Probe with High Selectivity toward Vascular Endothelial Cells for and beyond Noninvasive Two-Photon Intravital Imaging of Brain Vasculature[END_REF] the presence of the water-solubilizing chains do not suffice to avoid molecular aggregation of (E/Z)TT P at high water contents, and the molecules self-assemble into nano-aggregates.

Similar evolution patterns are observed for the AIE properties of the pure (E) and (Z) isomers of TT P , although precipitation onset is shifted to fH2O ca 60%. At fH2O of 90%, the fluorescence quantum yields of (Z) and (E) isomers reach a maximal Φf = 0.38 and 0.37 respectively. These values, similar to those observed with the (E/Z) mixture of TT P , are however significantly lower than the values reached in their parent stereopure monomeric analogues in nano-suspensions. [START_REF] Rouillon | Two-Photon Absorbing AIEgens: Influence of Stereoconfiguration on Their Crystallinity and Spectroscopic Properties and Applications in Bioimaging[END_REF] This similarity of AIE properties between the stereopure versions and the mixture of (E/Z)TT P thus suggests that the nature of the supramolecular assemblies formed in the case of PHEA chain chromophores does not markedly depend on the stereochemistry of the chromophores. This strikingly differs from our recent observation on similar AIEgens devoid of PHEA side substituents, where the higher crystallinity of the stereopure sample clearly benefited the AIE. In other words, this suggests that, quite expectedly, the presence of the PHEA chain influences the chromophore packing in the nano-assemblies.

NMR study of (E/Z)TT P

To gain understanding on the nature of the interactions between (E/Z)TT P polymers as a function of fH2O, and the characteristic size of their assemblies, NMR experiments were then performed in different D2O/ DMSO-d6 mixtures.

As a first notice, 1 H NMR spectra reveal a peculiar evolution of the signal belonging to the chromophoric part and the polymer chains upon increasing of fD2O (Fig. S15). Indeed, while the signals of the polymer chains remain constant in width and intensity, both the aromatic signals of the chromophore (between 6.80 and 7.45 ppm) and the signal of the two methyl end chains (1.24 ppm) broaden and decrease drastically with increasing the water fraction. At fD2O as low as 10%, these are already hardly distinguishable from the baseline and are no longer visible at fD2O = 50%.

It would thus seem that aggregation phenomena between the hydrophobic parts of the core (chromophore and attached initiator group) take place in an aqueous medium, in agreement with the spectroscopic evolutions observed. Again, this evolution contrasts strongly with previous examples of similar polymer appended chromophores reported by us, [START_REF] Pascal | Keto-polymethines: a versatile class of dyes with outstanding spectroscopic properties for in cellulo and in vivo two-photon microscopy imaging[END_REF] where the relative intensity of the different peaks was unchanged between DMSO-d6 and D2O media, characteristic of individual macromolecular objects in solution. Such a behavior is classically observed and has been studied in depth in the past during the self-aggregation process of conjugated polymer such as polythiophenes. [START_REF] Parenti | -Stacking Signature in NMR Solution Spectra of Thiophene-Based Conjugated Polymers[END_REF] It indicates here that, like in the latter, π-stacking constitutes unsurprisingly the main driving force for the self-assembly of the polymer chains.

To investigate this aggregation phenomenon, and its dependence on medium composition and chromophore stereoconfiguration, 2D diffusion-ordered NMR spectroscopy (DOSY) was then used (available as Supplementary Materials S3, Fig. S18-S29).

First, in pure DMSO-d6 solution, DOSY was used to verify that (E/Z)TT P molecules were present as individual polymer chains, and to determine their associated polydispersity index (Đ).

thanks to the methodology initially developed by Delsuc et al. and tested in the past on similar polymers. [START_REF] Viéville | Polydispersity index of polymers revealed by DOSY NMR[END_REF][START_REF] Appukuttan | Mild and efficient bromination of poly(hydroxyethyl acrylate) and its use towards ionic-liquid containing polymers[END_REF] In the case of all samples studied herein, the NMR signals attributed to (E/Z)TT P belong to the same polymer species (with a single diffusion constant, see considering our previous results. [START_REF] Massin | A water soluble probe with near infrared two-photon absorption and polarity-induced fluorescence for cerebral vascular imaging[END_REF][START_REF] Pascal | Keto-polymethines: a versatile class of dyes with outstanding spectroscopic properties for in cellulo and in vivo two-photon microscopy imaging[END_REF] In each solvent, diffusion coefficient values D of a molecule, macromolecule or nano-object relates to the gyration radius of the latter, considered as a random coil: thus, comparison of the D values obtained for the (Z) and (E) isomers of TT P featuring similar polymer side chain lengths (here, n=16) brings valuable information regarding the impact of stereochemistry on the characteristic dimensions of the associated coil. In DMSO-d6 solution, similar mean diffusion coefficients are monitored for the (Z) and (E) isomers (59 and 52 µm²/s respectively), characteristic of a comparable mobility and size for both solvated molecules (Fig. S19 andS20). As expected, the average diffusion coefficients D are smaller in D2O, as a consequence of the aggregation process, yet similar for each isomer (25 for the (E) isomer and 26 µm²/s for (Z) isomer, Fig. S21 and S22). This indicates a similar nature for the small-sized nano-objects formed by molecular aggregation in the case of both stereoconfigurations.

Wishing to get more insight into the aggregation processes taking place at high water contents, we then monitored in a next series of experiments the evolution of the D(E/Z)TT P coefficients as a function of fD2O (Fig. 4, Table S1). Since the spectroscopic studies had already underlined that the stereopurity had only little impact on the AIE properties, this study was limited to the (E/Z) mixture of TT P , available in larger quantities (as typically required for DOSY analyses) than its stereopure analogues. First, a global very noticeable decrease of D(E/Z)TT P values could be qualitatively observed upon increasing the deuterated water fraction from the initial pure DMSO-d6 sample, in agreement with the suspected aggregation process (Figs S23-S29). However, when comparing systems in different solvents compositions, diffusion coefficient of a given object does not solely depend on its radius, but also critically on the viscosity of the medium: it is well documented that the latter varies strongly in DMSO/water mixtures depending on the ratio of each solvent. In order to discriminate between both contributions, it is possible and beneficial to use a viscosity-corrected diffusion coefficient. Recent studies have indeed highlighted that, providing that polymer concentration was kept low in the medium (ca 1mg/mL), using such viscositycorrected values for the diffusion signal allowed a precise evaluation and comparison of macromolecules dimensions even in solvents possessing very different intrinsic viscosities. [START_REF] Nieto-Draghi | Transport properties of dimethyl sulfoxide aqueous solutions[END_REF] We thus considered in a next step the variations of a corrected diffusion coefficient defined, for each composition fD20 by equation 2:

𝐷 𝜂 = 𝐷 (𝐸/𝑍 )𝑇𝑇 𝑃 𝜂 𝜂 𝐷𝑀𝑆𝑂 (2)
With the viscosity η (mPa.s) of the DMSO/water mixture of the considered sample, [START_REF] Lebel | Density, Viscosity, Refractive Index, and Hygroscopicity of Mixtures of Water and Dimethyl Sulfoxide[END_REF] 𝜂 𝐷𝑀𝑆𝑂 the viscosity of pure DMSO.

Using this viscosity corrected diffusion coefficient we notice for fD20 > 10%, a progressive decrease in the corrected diffusion coefficient Dη as fD2O is increased (Fig. 4, Table S1). This confirms that a progressive aggregation process occurs, and that the average size of the resulting nanoaggregates gets bigger and reaches a maximal size in pure water, in agreement with the observed AIE evolution.

Although DOSY NMR brings conclusive evidence for the formation of nanoparticles by selfassembly of (E/Z)TT P at high water content, extrapolation of the characteristic size of the nanoobjects diffusion coefficient is inherently prone to error, specifically for non-rigid and nonperfectly spherical nano-objects. [START_REF] Hinton | Diffusion ordered 2D NMR spectroscopy of phospholipid vesicles: determination of vesicle size distributions[END_REF][START_REF] Canzi | Diffusion-Ordered NMR Spectroscopy as a Reliable Alternative to TEM for Determining the Size of Gold Nanoparticles in Organic Solutions[END_REF] In order to get additional and complementary insight on the dimension of the self-assembled nanoparticles, DLS experiments were thus undertaken on the aqueous suspensions of (E/Z)TT P (n=16). As expected, no scattering signal was obtained for the initial solution of the (E/Z) mixture of TT P in DMSO. In contrast, reliable DLS correlogram could not be obtained for both isomers (Fig. 5), with fH2O < 70%. Again, in contrast with what had been noticed in our previous work with the monomeric analogues (E/Z)TT OMe , where nanoprecipitation in water resulted in the formation of particles with a 150-200 nm average diameter, [START_REF] Rouillon | Two-Photon Absorbing AIEgens: Influence of Stereoconfiguration on Their Crystallinity and Spectroscopic Properties and Applications in Bioimaging[END_REF] an homogeneous suspension of small nanoparticles was observed here: for fH2O = 70%,

Biological studies

In a previous report, we showed that suspensions of non-polymeric (E/Z)TT OMe were efficient in providing highly contrasted two-photon fluorescence microscopy images, both in cellulo and in vivo (after injection in the caudal vein of zebrafish embryos). However long-term following of the fate of the zebrafish embryos revealed that various malformations and premature death occurred within the days after injections, raising important issues regarding the biocompatibility of these nano-objects. Size and surface functionalization of nanoparticles are indeed two important parameters that largely determine their fate and biocompatibility in living systems, both of which turned out to be suboptimal in this initial works.

Since our present study allowed us to underline several promising features of (E/Z)TT P as potential two-photon luminescent contrast agent, we decided to further investigate that point using similar setups and biological systems as in our abovementioned work. More specifically we aimed at establishing whether the adjunction of polymer side substituents and the resulting impact on the features of the self-assembled nano-systems could be beneficial in terms of biocompatibility.

In a first step, we studied the biocompatibility of such compounds on human cells in culture.

The goal was to determine which maximal concentration could be used while avoiding any significant cell death. To that end, increasing concentrations of compound in its isomeric (Z) and (E) form, were incubated with MCF-7 cells for 3 days. The result reported in figure 7a suggested that, even at such high concentrations as 150 µg.mL -1 , both compounds were safe for the cell. In figure 7b, we can see that both compounds are highly luminescent (green dots) and efficiently internalized in the cancer cells, which plasmatic membranes are stained with cell mask (in red). In this experiment, isomers in living cells were revealed by two-photon excitation at 750 nm excitation wavelength. This imaging potential seems very similar to those obtained in our previous report on (E/Z)TT OMe . Then, to determine the possibility for biomedical use, the imaging potential was analyzed in vivo, in zebrafish embryos. After injection in the caudal vein, TT P , either in its E or Z form, provide high luminescence signal visible under UV-light excitation (pseudocolor green, Fig. 8a) and exhibits a high solubility and bioavailability in all the organism. Importantly, the luminescence of these compounds was also investigated in two-photon microscopy conditions under pulsed laser excitation (Fig. 8b) strong capability of imaging was observed when excited at 750 nm, similar to that achieved in our aforementioned report. [START_REF] Rouillon | Two-Photon Absorbing AIEgens: Influence of Stereoconfiguration on Their Crystallinity and Spectroscopic Properties and Applications in Bioimaging[END_REF] In parallel, the biocompatibility of both isomers injected in the blood stream of embryos was studied. For 3 days, embryos development, rate heart and their mobility were daily monitored. The data reported in the table (Fig. 8c) demonstrate for both isomers that no toxicity could be detected in zebrafish embryos in the studied conditions, translating into no increase in the mortality of the nL of TT P (isomer Z or isomer E in their pure form, n=16) at 1 mg.mL -1 . One hour after injection, embryos were observed using a) one-photon excitation microscopy at 340-380 nm and b) twophoton excitation microscopy at 750 nm. c) Embryos were then maintained 3 days in breeding conditions to analyze every day the biocompatibility of Z isomer and E isomer according to the parameters reported in the table, with "+" for presence and "-" for absence.

CONCLUSION

In this paper, we illustrate the benefits in functionalizing TPE based AIEgens, which structure is specifically optimized for two-photon induced emission properties at the solid state, with hydrophilic side polymer chains. In DMSO, the resulting bolaamphiphilic macromolecular chromophores (E/Z)TT P exist as individual, low-emissive (φf ≈ 0.07) polymer chains. This is attested by DOSY NMR experiments, which indicate that, for both the (Z) and (E) isomers, a similar length of the polymer arms (n = 16 units) and a similar low polydispersity (Đ = 1.22) can be achieved, in agreement with the controlled nature of the radical polymerization.

In contrast, when dispersed in water, either from nano-precipitation of a concentrated solution of the molecule in DMSO or by direct mixing of the polymer powder in pure water or physiological solution, a characteristic AIE phenomenon is observed which correlates to the formation of small nano-bundles of several chromophore units of (E/Z)TT P . DLS analyses carried out on both the (Z) and (E) isomers confirm the presence of nano-aggregates at fH2O > 0.5, with a mean size ca.

Figure 1 .

 1 Figure 1. Chemical structure of (E/Z)TT P and its precursors.

  photon absorption (1PA) induced fluorescence spectra.

Figure 2 .

 2 Figure 2. a) Absorption (dashed) and emission (solid lines) spectra of the (E)-isomer (orange) and

Figure 3 .

 3 Figure 3. Emission of a) the isomers mixture, b) the Z-isomer and c) the E-isomer of TT P (n=16)

  Fig S18 for a representative case where n=16). Polydispersity index determination provides a value for Đ = 1.22 for (E/Z)TT P with n=16 which, although in the high range of what is usually achieved by ATRP, appears satisfactory

  d = 25 nm (PDI = 0.29) and fH2O = 90%, d = 26 nm.(PDI = 0.28; Fig.5). Then, upon simple mixing of the dry polymeric materials in pure distilled water, this average size is halved (around 10 nm for (E/Z)TT P ; PDI = 0.42) and slightly larger nano-suspensions are observed for the (E) isomer compared to (Z) (12 vs. 10 nm, PDI=0.46 vs PDI=0.42, Fig.5, Figs S31-S32). This result indicates that, as expected, the bolaamphiphilic nature of our polymer appended chromophore favors the controlled formation of small and monodisperse nano-objects, which size seems ideally suited for bio-related applications. This was further verified by performing low-resolution TEM measurements on (E/Z)TT P . Although the obtained images present only limited contrast because of the pure organic nature of the studied compound, they confirm the formation of spherical organic nanoparticles with an average diameter ca 10-15 nm, in good agreement with what had been calculated through both DOSY and DLS measurements (Fig.5c, Fig S34-36).

Figure 5 .

 5 Figure 5. DLS of a) (E/Z) mixture of TT P in DMSO/water mixtures and b) stereopure TT P in

Figure 6 .

 6 Figure 6. Plot of the evolution of the measured surface tension vs logarithm of the concentration

Figure 7 .

 7 Figure 7. In cellulo studies a) Cytotoxic study of E and Z isomers of TT P (n=16). Increasing concentrations (from 2.5 to 150 µg.mL -1 ) of compounds were incubated with MCF-7 cells for 3

  said embryos. Survival and development of the zebrafish embryos, as monitored over several days, are indeed perfectly similar in the treated and control embryos. These results constitute a marked improvement in comparison with our recently published results with nano-precipitated assemblies of TT OMe chromophores and highlight the strong potential of TT P for long term bioimaging of living organisms.

Figure 8 .

 8 Figure 8. In vivo studies. Casper zebrafish embryos of 72 hpf were intravenously injected with 10

10- 15

 15 ABBREVIATIONSATRP, Atom-Transfer Radical Polymerization; CDCl3, deuterated chloroform; DLS, Dynamic

Table 1 .

 1 Spectroscopic properties of TT P isomers in solution in DMSO.

		(Z)TT P	(E)TT P
	λabs (nm)	357	374
	λem (nm)	480	477
	Stokes shift (cm -1 )	7200	5800
	Φf	0.025	0.018
	σ2PA (GM) [λmax (nm)]	140 [710]	100 [710]

Stokes-Einstein equation relates the diffusion coefficient D of a well-defined spherical nanoobject to its radius r, according to equation (3):

Where kb is the Boltzmann constant (1.380 649 × 10 -23 J.K -1 ), T the temperature in Kelvins (here taken as 298 K) and η the dynamic viscosity, in Pa.s. The latter is well known and has been tabulated in the past for different DMSO/water mixtures. [START_REF] Lebel | Density, Viscosity, Refractive Index, and Hygroscopicity of Mixtures of Water and Dimethyl Sulfoxide[END_REF] Using these tabulated values, we calculated a nanoparticle radius (considered as rigid spheres) ca 7 nm in DMSO-d6:D2O, 70:30 (v/v, corresponding to the onset of the self-assembly process).

In pure water, it culminates to about 9 nm, i.e. a diameter of about 18 nm, in the spherical assumption.

Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM) studies

progressive restriction in the measured diffusion coefficient for chemical shifts belonging to the polymer chain upon increasing fH20, to reach its lowest value in pure water, confirming that a selfassembly process drives the formation of the fluorescent organic nanoparticles. In pure water, CMC of (E/Z)TT P is about 0.75 μM. It is thereby confirmed that the strategy introduced herein is efficient in yielding ultra-bright, two-photon active organic nanoparticles with features optimized in regard with the target biological applications.

To illustrate the benefits of our approach in the specific framework of intravital bioimaging, the resulting aqueous suspension of both (Z) and (E) TT P (fH20 = 100%) were used as nano-sized fluorescent organic nanoparticle contrast agents in epifluorescence and two-photon fluorescence microscopy of the vascular systems of zebrafish embryos. Besides the favorable spectroscopic properties of the nano-objects, which afforded in all cases highly contrasted images, a distinctive advantage of the polymer functionalized two-photon AIEgens reported herein in comparison with their molecular analogues is their much-improved biocompatibility, which enables longer term studies on living systems and might constitute a keystone and determining feature of this strategy towards its possible future use in a biomedical context.

ASSOCIATED CONTENT

Supporting Information. Detailed synthetic procedures; hard-copies of 1 H and [START_REF] Bui | Near infrared two photon imaging using a bright cationic Yb(iii) bioprobe spontaneously internalized into live cells[END_REF] C NMR spectra for all new molecules and polymers; conditions for separation of chromophoric macroinitiators by preparative HPLC on chiral stationary phases and associated chromatograms; complementary fluorescence studies (chart) regarding the dependence on fluorescence quantum yield to polymer length; hard-copies of all DOSY NMR spectra used in the study; pictures of 2.10 -5 M (E/Z)TT P in different DMSO/H2O mixtures (fH2O from 0% to 100%); surface tension measurements data; additional TEM images.

AUTHOR INFORMATION

Corresponding Author

magali.gary-bobo@inserm.fr, cyrille.monnereau@ens-lyon.fr; Present Addresses †Department of Chemistry and Chemical Engineering, Chalmers University of Technology, Göteborg 412 96, Sweden

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Funding Sources

The imaging facility MRI is a member of the national infrastructure France-BioImaging supported by the French National Research Agency (ANR-10-INBS-04, «Investments for the future»).


Lee Osterhout
email: losterho@u.washington.edu

Andrew Poliakov

Kayo Inoue

Judith Mclaughlin

Geoffrey Valentine

Ilona Pitkanen

Cheryl Frenck-Mestre

Julia Hirschensohn

Second-language learning and changes in the brain

Keywords: Second language, plasticity, ERPs, N400, P600, VBM, language processing

Presumably, second-language (L2) learning is mediated by changes in the brain. Little is known about what changes in the brain, how the brain changes, or when these changes occur during learning. Here, we illustrate by way of example how modern brain-based methods can be used to discern some of the changes that occur during L2 learning.

Preliminary results from three studies indicate that classroom-based L2 instruction can result in changes in the brain's electrical activity, in the location of this activity within the brain, and in the structure of the learners' brains. These changes can occur during the earliest stages of L2 acquisition.

Introduction

Experience can change both the function and the structure of the brain (Münte, Altenmüller, & Jäcke, 2002;[START_REF] Van Praag | Neural consequences of environmental enrichment[END_REF]. Like other experiences, the experience of learning a second language (L2) is presumably accompanied by changes in the brain. It seems reasonable to presume that how, when, and where these changes occur is relevant (and possibly even essential) to a truly compelling understanding of L2 acquisition. At present, however, almost nothing is known about what changes in the brain during L2 learning, when these changes occur, and how they reflect L2 learning.

Fortunately, the current era is one of rapid methodological innovation with respect to non-invasive measurement of the human brain. The question of interest is whether these modern methods can detect changes in the brain that occur with L2 acquisition.

Here, we describe preliminary results from ongoing experiments showing that these methods might in fact be sensitive to some of the brain changes that occur during L2 acquisition. Our preliminary data suggest these methods might be sensitive to changes over time in the brain's electrophysiological response to L2 stimuli, changes in the neural sources of that electrical activity, and even changes to the structure of the brain itself.

Most of the work reviewed below involves longitudinal studies of novice, Englishspeaking L2 learners progressing through their first years of university-based L2 instruction. It seems likely, therefore, that the changes we report here are relevant to the classroom settings that typify L2 instruction in the United States and many other countries.

Changes in the brain's electrophysiological response to L2 stimuli

Aspects of the brain's electrophysiological activity can be recorded non-invasively from the scalp. For example, event-related brain potentials (ERPs) reflect synchronized postsynaptic activity in cortical pyramidal neurons. In our laboratory, we have used ERPs to track learning-related changes in brain function. In particular, we have examined the rate at which L2 knowledge is incorporated into the learner's on-line, realtime language comprehension system. To achieve this goal, we have recorded ERPs while learners read or listen to tokens of the L2 [START_REF] Mclaughlin | Neural correlates of second-language word learning: minimal instruction produces rapid change[END_REF][START_REF] Osterhout | Novice learners, longitudinal designs, and event-related potentials: A paradigm for exploring the neurocognition of second-language processing[END_REF]. Our work has primarily involved longitudinal studies that assess changes in the brain response to L2 sentences that occur during the earliest stages of L2 learning. This approach was motivated by prior work showing that certain linguistic manipulations elicit robust effects in the ERP. The crucial finding has been that syntactic and semantic anomalies elicit qualitatively distinct ERP effects, and that these effects are characterized by distinct temporal properties. Semantic anomalies (e.g., The cat will bake the food …) elicit a negative wave that peaks at about 400 ms after the anomalous word appears (the N400 effect; Fig. 1A) [START_REF] Kutas | Reading senseless sentences: Brain potentials reflect semantic anomaly[END_REF][START_REF] Osterhout | On the distinctiveness, independence, and time course of the brain responses to syntactic and semantic anomalies[END_REF].

By contrast, syntactic anomalies (e.g., The cat will eating the food …) elicit a large positive wave that onsets at about 500 ms after presentation of the anomalous word and persists for at least half a second (the P600 effect; Fig. 1B) [START_REF] Osterhout | Event-related brain potentials elicited by syntactic anomaly[END_REF][START_REF] Osterhout | Event-related potentials and syntactic anomaly: Evidence of anomaly detection during the perception of continuous speech[END_REF][START_REF] Osterhout | Event-related brain potentials elicited by failure to agree[END_REF][START_REF] Osterhout | On the distinctiveness, independence, and time course of the brain responses to syntactic and semantic anomalies[END_REF]. In some studies, syntactic anomalies have also elicited a negativity over anterior regions of the scalp, with onsets ranging from 100 to 300 ms [START_REF] Friederici | The time course of syntactic activation during language processing: a model based on neuropsychological and neurophysiological data[END_REF][START_REF] Osterhout | Event-related brain potentials elicited by syntactic anomaly[END_REF].

_________________________________

Insert Fig. 1 about here _________________________________ These results suggest that separable syntactic and semantic processes exist. In an L2 learning context, one implication is that L2 learners must somehow segregate linguistic input into those aspects of the language that relate to sentence form and those that relate to sentence meaning. That is, learners "grammaticalize" some aspects of the L2, but not others. In our work, what we mean by "grammaticalization" is specifically the instantiation of grammatical knowledge into the learner's on-line, real-time language processing system. Our assumption is that, once a feature of the L2 has been grammaticalized, violations of that aspect of the grammar should elicit a P600 effect.

To investigate grammaticalization during L2 learning, we have focused on the acquisition of grammatical features and their associated morphosyntactic rules. These features (and how they are involved in morphosyntax) vary across languages. For example, English and French both have sentential agreement (i.e., agreement of the verb with the subject in verbal person and number; e.g., I like vs. He likes), but only French has noun phrase agreement, that is, agreement between the noun and its determiner/ adjective in number and gender (e.g., le garçon vs. les garçons 'the-masc-sg boy, the-pl boys', excluding the restricted English case of this/those/these).

What factors might inhibit or facilitate grammaticalization of these features and their morphosyntactic rules? One frequent claim is that only grammatical features that are present in the L1 can be acquired during L2 acquisition [START_REF] Hawkins | Explaining the acquisition and non-acquisition of determiner-noun gender concord in French and Spanish[END_REF]. Other researchers argue that novel L2 features can be learned, albeit more slowly than those that are present in the L1 [START_REF] White | Second Language Acquisition and Universal Grammar[END_REF]. Thus, there is no consensus about whether, or when during acquisition, L2 learners acquire L2 features and morphosyntactic rules that are not present in their L1.

Another factor that seems likely to play a role in L2 grammatical morpheme learning is the covariation between morphology and phonology. For example, French has an opaque orthography due to many suffixes being phonologically silent. Thus, the plural suffix -s, which marks the plural orthographically across all elements in the NP (le-s jeune-s fille-s 'the young girls') is silent on the noun, determiner, and adjective in almost all instances. A similar situation arises in the verb phrase (VP), where variations in verbal person are marked orthographically on the verb but are silent in most oral forms. Thus the different inflections for a regular verb such as marcher (to walk) in present tense sound identical across four different persons/spellings. The effect of the 'missing' phonological cue is notorious on spelling. Errors such as les chien or Ils mange are frequent [START_REF] Negro | Subject-verb agreement with present and imperfect tenses: a developmental study from 2 nd to 7 th grade[END_REF], and are often made by native-Frenchspeaking adults as well as by children. Such errors are much rarer when phonology is available as a cue [START_REF] Largy | Oral cues improve subject-verb agreement in written French[END_REF].

Given this evidence, a reasonable prediction is that L2 learners will acquire an L2 feature or morphosyntactic rule more quickly when the relevant inflectional morphology is phonologically realized. However, this possibility has received little direct attention in the recent L2 literature. It also seems likely that L1-L2 similarity and phonologicalmorphological covariation might have interactive effects during L2 learning. For example, L1-L2 similarity combined with phonological realization of the relevant grammatical morphemes might lead to very fast learning, whereas L1-L2 dissimilarity combined with no phonological realization might lead to very slow learning.

We investigated these predictions using a longitudinal experimental design involving 14 English-speaking novice French learners progressing through their first year of French instruction at the University of Washington 1 . Our stimuli were as follows:

(1) Sept plus cinq\?livre font douze.

'seven plus five/book make twelve' semantic condition

(2) Tu adores\*adorez le français.

'you-2-sg adore-2-sg \ adore-2-pl the French' verbal person agreement condition/phonologically realized (3) Tu manges des hamburgers\*hamburger pour diner.

'you-2-sg eat-2-sg some-pl hamburgers-pl \ hamburger-sg for dinner' number agreement condition/phonologically unrealized In (1), the noun livre is semantically anomalous. In (2), the verb adorez is conjugated incorrectly, given the preceding sentence fragment. In (3), the noun hamburger disagrees with the syntactic number of the plural article. Our stimuli were selected from the material in the textbook assigned during the first month of instruction. The anomalous items in the verbal person condition involved a grammatical rule that was present in the L1 and an orally realized contrast between inflectional morphemes. The anomalous items in the number agreement condition involved a rule that was not present in the L1 and a phonologically unrealized contrast between inflectional morphemes. Therefore, our prediction was that L2 learners would respond to the anomaly in (2) with less L2 exposure compared to the anomaly in (3).

For each condition, subjects read 30 exemplars each of anomalous and wellformed control sentences. Sentences were counterbalanced across lists, such that each subject saw only one version of a particular sentence frame. The 180 sentences in each list were pseudorandomly ordered prior to presentation. Sentences were presented wordby-word on a computer screen, with each word being presented for 350 ms and with a 300 ms blank-screen interval between words. The final word in each sentence was followed by a 1450-ms blank screen interval, after which the subject was prompted to make a "sentence acceptability" judgment about the preceding sentence. Continuous EEG was recorded from 13 scalp sites and averaged off-line.

As expected, the native French speakers showed an N400 effect to the semantically anomalous words (1) and large P600 effects to the two types of syntactic anomalies (2)-(3). The learners, as is often the case, showed striking individual differences, both in a behavioral "sentence acceptability judgment" task and in the pattern of ERPs elicited by the anomalous stimuli. We segregated the learners into upper ("fast learners", n = 7) and lower ("slow learners", n = 7) halves, based on their performance in the sentence-acceptability judgment task (mean d-primes for the session 3 sentence acceptability judgments, averaged over the three conditions, were 2.7 and 1.5 for the fast and slow learners, respectively). ERPs were then averaged separately for each group.

Results for the "fast learner" group will be described here. At each testing session, including the initial session that occurred after just one month of instruction, semantically anomalous words elicited a robust N400 effect (midline electrodes: F(1,6) = 24.61, p < .003), and this effect changed minimally with increasing instruction (d-primes for sentence-acceptability task were 2. 0, 3.0, and3.1 for sessions 1, 2, and3). Results for the verbal person agreement condition are shown in Fig. 2. After just one month of instruction, the learners' brains discriminated between the syntactically well-formed and ill-formed sentences (midline electrodes: F(1,6) = 5.58, p = 0.05). However, rather than eliciting the P600 effect (as we saw in native French speakers), the syntactically anomalous words elicited an N400-like effect. (This effect did not differ in distribution from the N400 effect elicited by the semantically anomalous words.) By four months, the N400 effect was replaced by a P600-like positivity (midline electrodes: F(1,6) = 8.73, p < 0.03; d-primes were 2.0, 3.5, and 3.5 for sessions 1, 2, and 3). Results for the nominal number agreement condition can be summarized easily: Learners performed very poorly in the sentence acceptability judgment task for these materials (d-prime = 0.5, 1.5, 1.6 for sessions 1,2, and 3), and there were no robust differences in the ERP responses to the agreeing and disagreeing stimuli. _________________________________ Insert Fig. 2 about here _________________________________ These results are consistent with the predictions we were testing. First, L1-L2 similarity combined with phonological realization of the relevant grammatical morphemes produced very fast L2 syntactic learning, whereas L1-L2 dissimilarity combined with no phonological realization produced very slow learning. This occurred even though our learners were drilled repeatedly on both rules from nearly the first day in class. However, the two rules we tested represent the ends of a putative continuum of morphosyntactic difficulty; without additional data it is impossible to know whether L1-L2 similarity or phonological realization of grammatical morphemes had a larger impact on the learning rate.

We also observed a discontinuous pattern over time in the response to the verbal person anomalies: early in learning, these anomalies elicited an N400 effect in learners, whereas later in learning these same anomalies elicited a P600 effect. Our hypothesis is that our learners were progressing through discrete stages of syntactic learning: They began by memorizing particular combinations of words and morphemes, and only later induced general syntactic rules [START_REF] Myles | Rote or Rule? Exploring the Role of Formulaic Language in Classroom Foreign Language Learning[END_REF]Wray, 2002;see Tomasello, 2000, for evidence that children go through similar stages during L1 acquisition). To be specific, an L2 learner might initially memorize the fact that certain subjects are followed by certain forms of the verb, without decomposing the verb into root + inflection or applying a general morphosyntactic agreement rule. In this stage of learning, the learner associates meanings with the undecomposed chunk of language, and either memorizes the two words as a chunk or learns about word sequence probabilities (e.g., that Tu 'you-2-sg' is followed by marches 'walk-2-sg', whereas Ils 'they-3-pl' / Nous 'we-1-pl' is followed by marchent 'walk-3-pl' / marchons 'walk-1-pl'). Violations of the verbal person rule (e.g., tu *adorez) result in novel word combinations, and hence elicit an N400 effect.

After more instruction, learners induce a general verbal person rule (tu -s, nous -ons, vous -ez, etc); violations of the rule elicit a P600 effect. If our interpretation is correct, then our adult L2 learners grammaticalized this aspect of the L2 after just a few months (~80 hours) of L2 instruction.

Changes in brain sources

Ideally, one would like to discern the neural sources of these language-sensitive ERP effects. By doing so, one might be able to describe changes in the brain across both time and space. Unfortunately, the source of a given ERP effect (the "inverse solution") cannot be known with certainty. This follows from the fact that a large number of source configurations could produce an identical pattern of activity across the scalp [START_REF] Nunez | Electric fields of the brain[END_REF]. Nonetheless, source estimates are possible given certain limiting assumptions. The traditional approach has been to search for point dipole sources [START_REF] Hämäläinen | Realistic conductivity geometry model of the human head for interpretation of neuromagnetic data[END_REF][START_REF] Henderson | The localization of equivalent dipoles of EEG sources by the application of electrical field theory[END_REF]Kavanaugh et al., 1978). In general, this entails assuming a small number of dipole sources and iterating through all possible combinations of dipole location, orientation, and strength, looking for the best match between the source model and the observed scalp distribution. This method brings with it numerous limitations and caveats [START_REF] Halgren | N400-like magnetoencephalography responses modulated by semantic context, word frequency, and lexical class in sentences[END_REF].

More recently developed alternative methods provide a tomographic analysis analogous to that provided by neuroimaging methods, but with vastly superior temporal resolution (and reduced spatial resolution; [START_REF] Michel | Spatiotemporal EEG Analysis and Distributed Source Estimation in Presurgical Epilepsy Evaluation[END_REF]. These methods, known as distributed source models, provide a linear inverse solution (unlike the traditional nonlinear dipole approach) to estimate the current distribution throughout the entire three-dimensional cortex. But in order to reach a unique solution, these models require assumptions that might not be valid. If the assumptions are not valid, then various errors can occur: simultaneously active areas can be missed, spurious sources can be generated, and the results can be excessively blurred (for extensive discussion, see [START_REF] Michel | Spatiotemporal EEG Analysis and Distributed Source Estimation in Presurgical Epilepsy Evaluation[END_REF].

In our lab, we have been using one type of distributed source model, Low Resolution Electromagnetic Tomography (LORETA; Pascual-Marqui, [START_REF] Pascual-Marqui | Low resolution electromagnetic tomography: a new method for localizing electrical activity in the brain[END_REF], to estimate the sources associated with processing sentences in the L1 and L2. The primary assumption of LORETA is that dramatic changes in current do not occur across contiguous areas of cortex (i.e., in adjacent voxels). In practice, this assumption often produces an excessively blurred solution. Because in our study the L1-L2 contrast is within subject, any distortions that result from violations of the smoothness assumption should be relatively constant across languages. Therefore, differences in the source distribution associated with processing sentences in two languages might be meaningful (or at least roughly indicative) even if the exact location of the sources is somewhat distorted.

In an ongoing study, we are recording ERPs from native English-speaking university students who are enrolled in L2 (French) instruction. Presently, we have recorded ERPs from 22 students who were approximately halfway through their second year of French instruction. The stimuli were sentences in the "semantic" and "verbal person agreement" conditions in the L2 ERP experiment described above, plus approximate English translations of the French sentences. Counterbalancing was used such that each subject saw only one version of each sentence across well-formed vs. illformed contrasts and L1vs. L2 contrasts. The methods were identical to those described, except that subjects were tested once with L1 (English) stimuli and once with L2 (French) stimuli; the order of languages was counterbalanced across subjects. Subjects were tested in 1 session lasting between 2 and 3 hours, with a break between the L1 and L2 lists. EEG was recorded from 61 channels rather than 13; the greater spatial sampling is crucial for better source estimates. As expected, the semantically and syntactically anomalous words elicited N400 and P600 effects, for both English (L1) and French (L2) (midline electrodes: semantic, F(1,19) = 9.02, p <.01; syntactic L1, F(1,19) = 47.54).

Interestingly, the P600 effect was much larger for the L1 than the L2 (F(1,19) = 5.28, p < .04), whereas the N400 effect was similar across the two languages (F(1,19) = 1.91, p > 1.8).

We then used LORETA to estimate the current distribution associated with normal sentence processing (rather than the processing of anomalous sentences, which has been the strategy in previously published work) within two critical time windows: the window associated with the N400 component (during which the brain is most robustly sensitive to conceptual aspects of the stimulus) and the window associated with the P600 effect (during which the brain is most robustly sensitive to syntax). LORETA analyses on the critical words in the well-formed verbal person agreement condition are shown in Figure 3. Two solutions are shown for each word, for representative time points in the N400 (300 to 500 ms) and P600 (500 to 800 ms) windows. For English (L1) stimuli, the LORETA solutions indicated a posterior distribution for the N400 window (including temporoparietal and extrastriate regions), and an anterior distribution for the P600 window (especially the left inferior frontal gyrus). The LORETA solutions for the same critical words in the French (L2) sentences are shown on the right half of Figure 3; clusters of current density are listed in Table 1. The L2 LORETA solutions in the N400 window were similar to those for the L1 stimuli, with a posterior current distribution for both nouns and verbs. However, the L2 LORETA solution in the P600 window were quite different from the L1 solutions; current density was greatest in the medial dorsal frontal lobe, rather than in the inferior frontal gyrus. Any interpretation would be premature; nonetheless, it is interesting to note that posterior lesions involving the left temporoparietal region often produce a deficit in processing word meanings, whereas lesions to the left inferior frontal lobe often produce agrammatic symptoms. Perhaps L2 learners quickly incorporate aspects of L2 word meaning into their on-line processing system, and engage similar neural circuits that access and integrate these meanings. L2 syntactic rules might take longer to incorporate in this way. One question we hope to answer in this longitudinal design is how much L2 experience is needed before the LORETA solutions become more like the L1 responses (e.g., the point at which L2 verbs engage the "anterior processing stream"). In additional experiments involving first-and third-year French students, our preliminary results indicate that the L2 solutions might become progressively more like the L1 solutions, across the first 3 years of L2 instruction.

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Changes in brain structure

Anatomical neuroimaging methods can be used to study the effects of learning on brain structures in vivo. These methods include morphometric and volumetric techniques that measure certain parameters of well-defined brain structures (for example, hippocampal volume or cortical thickness). More recently, a number of unbiased, whole-brain techniques have been developed. One such technique is voxel-based morphometry (VBM; [START_REF] Mechelli | Voxel-based morphometry of the human brain: Methods and applications[END_REF]. In its simplest application, VBM involves a voxel-by-voxel comparison of the relative concentration of gray matter (GM) and white matter (WM). This is achieved using whole-brain MRI scans.

VBM was originally devised to examine structural abnormalities in patients.

Recent work using neurologically normal subjects, however, has shown that VBM can reveal the effects of learning and practice on brain structure. For example, [START_REF] Maguire | Navigation-related structural change in the hippocampi of taxi drivers[END_REF] used VBM to test whether structural changes could be detected in the brains of London taxi drivers as a result of extensive experience with spatial navigation. The VBM data indicated that the posterior hippocampi of taxi drivers were significantly larger than those of healthy controls. Furthermore, hippocampal volume correlated with the amount of time spent as a taxi driver. These results suggest that the posterior hippocampi may expand regionally in individuals who have extensive experience with spatial navigation. However, an alternative explanation could attribute this result to selection rather than plasticity (e.g., people with larger posterior hippocampi might gravitate to spatially demanding occupations). Longitudinally designed VBM studies can help reduce such uncertainties. For example, [START_REF] Draganski | Neuroplasticity: changes in grey matter induced by training[END_REF] demonstrated that acquisition of new skills may indeed change GM density. Brain scans were acquired from healthy subjects before they learned juggling and 3 months later when they had become skilled performers. The comparison of the scans acquired before and after practice revealed an expansion in gray matter in bilateral mid-temporal areas and left posterior intra-parietal sulcus. These findings were specific to the training stimulus, as a group of controls showed no changes in gray matter over the same period. More recently, Draganski and colleagues [START_REF] Draganski | Temporal and spatial dynamics of brain structure changes during extensive learning[END_REF] described GM density changes in a group of medical students studying for their final examination. Although these findings are intriguing, the mechanism of changes detected by VBM is still poorly understood, particularly in healthy subjects. Methods other than whole-brain MRI will be required to investigate whether the gray-matter increases induced by the acquisition of new skills are related to changes in neuropil, neuronal size, and/or dendritic or axonal arborization.

Just a few studies have used VBM to investigate possible effects of language learning on brain structure. [START_REF] Mechelli | Neurolinguistics: structural plasticity in the bilingual brain[END_REF] demonstrated that systematic measurable structural changes are present in the brains of individuals who have learned multiple languages, L2 learners' brains, and that the degree of structural variation depends on the age of acquisition of the second language and the proficiency attained (as well as on the number of languages acquired). Golestani et al. (2006) described anatomical correlates of learning novel speech sounds studied using sulcul anatomy and VBM of white as well as gray matter. Although the results from these VBM-and-language studies are quite interesting, they have employed cross-sectional designs. Cross-sectional VBM designs introduce certain problems of interpretation (for discussion see [START_REF] Bookstein | Voxel-based morphometry should not be used with imperfectly registered images[END_REF], Ashburner & Friston, 2001[START_REF] Mechelli | Neurolinguistics: structural plasticity in the bilingual brain[END_REF], and also uncertainty as to whether VBM differences are due to "learning" or to "preselection."

In our lab, we have completed a longitudinal VBM pilot study of L2 learning.

Our four subjects were university students enrolled in an intensive first-year Spanish course. The course lasted nine weeks and met daily for 3 _ hours of instruction. In our longitudinal design, each learner participated in two MRI sessions of six MRI scans each.

Session 1 occurred after 20 or 21 days of instruction. Session 2 occurred between 1 day before and 3 days after the end of instruction. For each subject, approximately 5 weeks of L2 instruction intervened between Sessions 1 and 2. The data from the four subjects (48 structural T1 scans total) were subsequently preprocessed and analyzed. We used the SPM5 software package and the VBM5 toolkit (C. Gaser) that implements enhancements and modifications needed for more advanced VBM analysis techniques.

Due to the small sample size (n = 4), we did not expect to find (nor did we find) statistically significant changes in GM density using an anatomically open hypothesis (i.e. statistical analyses not constrained to any anatomical region). Instead, we used a region of interest approach based on the results of the [START_REF] Mechelli | Neurolinguistics: structural plasticity in the bilingual brain[END_REF] crosssectional VBM study of bilingual and multilingual speakers. Mechelli et al. report that the amount of experience with a second language correlates with GM changes in a lefthemisphere inferior parietal region (MNI coordinates: x = -48, y = -62, z = 44). In our VBM analysis, we used a Small Volume Correction with a spherical 10 mm region of search centered at the MNI coordinates reported by Mechelli et al. This analysis detected a cluster of significant (p < 0.05) GM density increase. The K E value reported for the cluster-level statistics of VBM was 373. This result suggests that structural changes resulting from learning a second language can be observed after a relatively short but intense instructional period, even in a small sample of learners. Of course, these results are preliminary and await replication in a larger-scale study.

Each method of investigation comes attached with cautions and limitations, and this is certainly true of VBM and related methods. For example, significant effects might be attributable to scanner or MR sequence differences rather than to the subjects themselves. Other potential issues relate to the difficulty of spatially normalizing brains, robustness of standard parametric tests and the interpretation of the results. Some apparently volumetric differences may be due to displacement without volume change.

Any factor which causes changes in voxel intensities in the original images (eg vascular changes, changes in hydration status leading to relaxation time changes, etc) may result in significant VBM differences.

One other limitation of VBM deserves particular mention: Tissue segmentation (into gray and white matter) is easier and more reliable in some parts of the brain than in other parts. Segmentation results will inevitably be affected by the presence of other tissues with similar contrast (for example, dura matter, large blood vessels etc.) and imaging artifacts (movement related artifacts including blood flow and eye movement artifacts, susceptibility artifacts near paranasal sinuses and ear canals and many others).

To illustrate this point, we assessed the variability of GM density maps across several scans within a session using data from our pilot VBM study described above.

Variability for one subject/session was estimated by computing the standard deviation of GM density maps estimated from each of the six scans of a single session. These were normalized and averaged to produce a map shown in Figure 4. This color-coded map is overlaid onto a GM probability density map. Yellow and red areas represent increased variability in GM density estimates. Figure 4 clearly indicates that variability is much higher in some parts of the brain (e.g., some regions in the temporal and parietal lobes) than in others; correspondingly, changes in GM density will be more observable where the variability is relatively low. _________________________________ Insert Fig. 4 about here _________________________________

Conclusions

Our results suggest that the brain of an adult second-language learner is a highly dynamic place, even during the earliest stages of L2 learning. Our results also suggest that modern methods are capable of revealing at least some of these changes. In particular, the methods used here are seemingly sensitive to changes in the brain's electrical activity, changes in the location of this activity within the brain, and changes in the structure of the learners' brains. Of course, our results are preliminary and await completion of the ongoing work, as well as subsequent replication.

At first glance, our results might seem surprising. A large literature has shown that the ability to learn a language seems to degrade with age; our learners were not young children but instead were young adults. The causes of age effects on L2 proficiency are controversial, but two frequently cited theoretical explanations stand out.

One explanation involves the putative existence of a critical period for L2 learning [START_REF] Johnson | Critical period effects in second language learning: The influence of maturational state on the acquisition of English as a second language[END_REF]. According to this explanation, language learning is constrained by maturational factors (specifically, brain maturation) that circumscribe a critical period (which by most accounts ends around puberty) for native-like attainment.

A second explanation attributes the age-related declines to the effects of increasing experience with a first language (Kuhl, 2004). Neural network simulations provide a convenient framework for understanding effects of L1 usage on L2 learning. In these simulations, early learning results in the entrenchment of optimal network patterns, after which new learning requires considerable training. Consistent with this view, it has been demonstrated that early experience with certain aspects of a first language (e.g., phonemes) seems to degrade the ability to learn aspects of a second language later in life (Kuhl, 2004).

Both of these explanations implicate the same underlying cause for age-related effects on language learning, namely, a reduction in neural plasticity that degrades the ability to learn and retain new linguistic information. However, despite the popularity of this view, there is little direct evidence to support it. The hypothesis also seems to be inconsistent with accumulating evidence that (at least in many respects) the brain remains remarkably plastic throughout much of life [START_REF] Van Praag | Neural consequences of environmental enrichment[END_REF]. The findings reported here are clearly too preliminary and insufficiently detailed to inform this debate in a substantive way. But these findings do allow us to hope that the methods in hand might, eventually, tell us something useful about the degree of neural plasticity in older learners and the role it plays in enabling and/or hindering L2 acquisition.

Table 1 .Fig 1 .

 11 Figure Captions

Fig. 2 .

 2 Fig. 2. ERPs to the French (L2) verbal person agreement condition, for fast learners. (a)

Fig. 3 .

 3 Fig. 3. LORETA solutions at 400 and 600 ms to critical words in the well-formed

Fig. 4 .

 4 Fig. 4. Variability in VBM signal. A color-coded map depicting heterogeneity in

  Fig. 1

A similar preliminary report of this experiment appeared in[START_REF] Osterhout | Novice learners, longitudinal designs, and event-related potentials: A paradigm for exploring the neurocognition of second-language processing[END_REF].

Acknowledgements

Preparation of this article was supported by Grants R01DC01947 and P30DC04661 from the National Institute on Deafness and Other Communication Disorders.