L Éa Chazot-Franguiadakis

Joelle Eid

Marius Socol

Bastien Molcrette

Philippe Gu Égan

Maryl Ène Mougel

Anna Salvetti

Fabien Montel
email: fabien.montel@ens-lyon.fr

Optical Quantification by Nanopores of Viruses, Extracellular Vesicles and Nanoparticles

Keywords: Nanopore, Virus, Extracellular Vesicles, Nanoparticles, Zero Mode Waveguide, Concentration measurement

Nanopores combined with optical approaches can be used to detect viral particles.

In this work, we demonstrate the ability of hydrodynamical driving and optical sensing to identify and quantify viral particles in a biological sample. We have developed a simple and rapid method which requires only fluorescent labelling of the particles and can therefore be applied to a wide range of virus type. The system operates in real time and at the single particle level while providing a low error on concentration (4%) and a low limit of detection of 10 5 particles/mL for an acquisition time of 60 seconds, with the ability to increase the acquisition time to achieve a lower limit.

Introduction

The rapid and accurate quantification of viruses is crucial to gain information on viral infection processes and is still a goal to achieve in clinical and basic research. Currently commercially available assays offer noteworthy detection limits, for example 200 DNA copies/mL for quantitative commercial real-time Polymerase Chain Reaction (qPCR)-based assays 1,2 , or 6 -10 × 10 5 particles/mL for Western Blot/ ELISA assays [START_REF] Coorssen | Quantitative femto-to attomole immunodetection of regulated secretory vesicle proteins critical to exocytosis[END_REF] . In any case, these methods are time-consuming (about 24 hours for ELISA or Western Blot), too specific (ELISA and Western Blot assays can be carried out only if a primary antibody against the protein of interest is available), and sometimes inaccurate (due to off-target effects and saturation of the signal) [START_REF] Ghosh | Accuracy of Western Blots[END_REF][START_REF] Eid | Advances in Continuous Microfluidics-Based Technologies for the Study of HIV Infection[END_REF] .Therefore, they do not fully respond to the requested specifications in several fields of application such as clinical diagnosis (live monitoring of virus levels in patient samples) and basic research which may focus on different stages of the virus cycle (entry into the cell, reverse transcription, release of new virions,...). To meet demand several microfluidic and single molecule based quantification techniques are now under development [START_REF] Eid | Advances in Continuous Microfluidics-Based Technologies for the Study of HIV Infection[END_REF][START_REF] Renner | Intact Viral Particle Counts Measured by Flow Virometry Provide Insight into the Infectivity and Genome Packaging Efficiency of Moloney Murine Leukemia Virus[END_REF][START_REF] Welsh | Towards defining reference materials for measuring extracellular vesicle refractive index, epitope abundance, size and concentration[END_REF] . For instance, commercial Nanoparticle Tracking Analysis (NTA) can be used to determine concentration and size distribution of fluorescent viral particles. NTA offers a limit of detection (LOD) of 10 6 particles/mL and a manipulation time of a few hours [START_REF] Kramberger | Evaluation of nanoparticle tracking analysis for total virus particle determination[END_REF][START_REF] Nikitin | Comparative Study of Non-Enveloped Icosahedral Viruses Size[END_REF][START_REF] Steppert | Quantification and characterization of virus-like particles by size-exclusion chromatography and nanoparticle tracking analysis[END_REF] . Interferometric light microscopy can also be used to quantify viruses while measuring scattered signal and Brownian motion but the LOD is elevated (10 8 -10 10 particles/mL) [START_REF] Roose-Amsaleg | Utilization of interferometric light microscopy for the rapid analysis of virus abundance in a river[END_REF] .

An interesting compromise between a low LOD and real-time acquisition can be found in nanopore devices that have been developed in recent years and have tended to become a new standard for DNA/RNA sequencing [START_REF] Workman | Nanopore native RNA sequencing of a human poly(A) transcriptome[END_REF][START_REF] De Coster | Towards population-scale longread sequencing[END_REF] . Nanopore devices with single-molecule and real-time detection capabilities are of great interest to detect and quantify viruses as they open new avenues for the development of simpler, faster and more compact quantification alternatives. Most methods based on nanopore rely on genome detection and quantification with electrical sensing [START_REF] Mcnaughton | Illumina and Nanopore methods for whole genome sequencing of hepatitis B virus (HBV)[END_REF][START_REF] Radukic | Nanopore sequencing of native adeno-associated virus (AAV) single-stranded DNA using a transposasebased rapid protocol[END_REF][START_REF] Nguyen Quang | Dynamic nanopore long-read sequencing analysis of HIV-1 splicing events during the early steps of infection[END_REF][START_REF] Stein | Quantification of mRNA expression using single-molecule nanopore sensing[END_REF] , but optical detection is a possible alternative. In that sense, Zero-Mode Waveguide (ZMW) methods combined with nanopore have been showed to be powerful tools, as they allow enhanced DNA sequencing [START_REF] Farhangdoust | Rapid Identification of DNA Fragments through Direct Sequencing with Electro-Optical Zero-Mode Waveguides[END_REF][START_REF] Spitzberg | Plasmonic-Nanopore Biosensors for Superior Single-Molecule Detection[END_REF][START_REF] Klughammer | Palladium zero-mode waveguides for optical singlemolecule detection with nanopores[END_REF] . Some other techniques using electrical detection, are probing the virus as a whole. These techniques, refered as Resistive Pulse Sensing techniques, are based on quantifying changes in resistance using a nanoscale constriction. In practice, a voltage is applied between the two sides of the pore and when an object enters the pore the variations of the ionic current are measured [START_REF] Harms | Single-particle electrophoresis in nanochannels[END_REF][START_REF] Harms | Monitoring Assembly of Virus Capsids with Nanofluidic Devices[END_REF][START_REF] Mcmullen | Stiff filamentous virus translocations through solid-state nanopores[END_REF] . It is thus possible to detect and quantify viruses with an interesting lower practical count limit of 5.10 7 particles/mL as reported by De Blois and Wesley team [START_REF] Deblois | Sizes and concentrations of several type C oncornaviruses and bacteriophage T2 by the resistive-pulse technique[END_REF] . Interestingly, these methods can be used to obtain information on the size of the objects, and even establish size distribution profile [START_REF] Vogel | Quantitative Sizing of Nano/Microparticles with a Tunable Elastomeric Pore Sensor[END_REF] , since the variation in resistance of the pore is directly related to the sizes of the pore and of the object crossing it. Nevertheless, to date they mainly focus on the determination of particle size and not on a quantitative measurement of concentration [START_REF] Yang | Quantification of virus particles using nanopore-based resistive-pulse sensing techniques[END_REF] . This can be explained by some limitations of these systems, notably the fact that they remain too unspecific since any object passing through the pore will be detected, as well as objects that do not cross the pore but only come close to the surface and diffuse back. This represents a major bottleneck in order to quantify viral concentration.

Moreover, it can be added that electrical detection can only detect particle transport in pores that are of the same diameter that particle which increases plugging risk. This is not the case, in optical detection, where the pore can be much larger than the virus size.

Ultimately, despite these advances, a technological gap remains as there is no nanoporeoptical technique to quantify virus concentration. It should be remembered that viruses are particular biological objects and whether for techniques using electrical or optical detection, the solid state nanopores are often made of SiN or SiC which makes the virus stick to the pore.

In this work, we proposed to use hydrodynamical driving of viral particles through artificial low cost nanopores and optical detection. We developed a simple and sensitive method which required only fluorescent labelling of particles and an initial calibration of our experimental setup. It allowed us to be more specific but also versatile. Moreover, the track-etched membranes that we used were particularly relevant for this type of application since they were optimized to reduce biofouling. We showed that our setup can be used to detect biological particles and accurately determine their concentration in a small volume of biological sample with a low LOD of about 10 5 particles/mL for an acquisition time of 60 seconds (possibility to increase the acquisition time over 60 seconds to reach a lower LOD, for example about 1 hour to reach a LOD of 10 3 particles/mL). Moreover, we detected different types of viral particles and also discriminated viruses from extracellular vesicles to finally shed light on the versatility of the method.

Results and discussion

The viral particles used in this study are representative of the large range of virus population often encountered either for biotechnology application, or as pathology generator: enveloped/non-enveloped, DNA, viruses, retroviruses as detailed in Fig. 1A. As enveloped viruses, Human Immunodeficiency Virus (HIV) was used as non-infectious Virus-Like Particle (VLP) form. VLP structurally mimics the native virus but is devoid of the viral genome.

It is a promising candidate for vaccination [START_REF] Cervera | Production of HIV-1-based virus-like particles for vaccination: achievements and limits[END_REF] . VLP HIV were obtained from monoclonal human HeLa cells stably expressing Gag-GFP. Murine Leukemia Virus (MLV) was used in the form of both VLP and complete enveloped virus cultivated from NIH3T3 mouse embryonic fibroblast cells. For these two retroviruses, fluorescent labelling relied on Green Fluorescent Protein (GFP, λ exc = 488nm/λ em = 507nm) insertion into viral genome and expressed as Gag-GFP fusion 28 , which did not alter the properties of the virus [START_REF] Voelkel | Protein transduction from retroviral Gag precursors[END_REF][START_REF] Mougel | NXF1 and CRM1 nuclear export pathways orchestrate nuclear export, translation and packaging of murine leukaemia retrovirus unspliced RNA[END_REF] .

As non-enveloped viruses we relied on Adeno-Associated Virus (AAV, serotypes 8/9) which have gained interest for genetic therapies and vaccination processes over past years.

Stocks of AAV-8 and AAV-9 recombinant particles were generated by calcium phosphate transfection of HEK-293 cells and analysed by AFM as described previously in litterature [START_REF] Salvetti | Factors influencing recombinant adeno-associated virus production[END_REF][START_REF] Carrasco-Salas | The extruded non-template strand determines the architecture of R-loops[END_REF] . Hepatite B Virus (HBV) was also used in a non-enveloped form (ie non-infectious form), that was purified from a viral stock of infectious HBV particles (genotype D) concentrated on a 20% sucrose cushion. AAV and HBV were fluorescently labelled using intercalating fluorophore YOYO-1 (Molecular Probes, 1mM in DMSO, λ exc = 491nm /λ em = 509nm ) which targets viral DNA.

Besides, we handled Extracellular Vesicles (EVs) which are constituted of lipids and proteins and sometimes contain cellular genetic material. Such composition often lead them to be mistaken for viruses. EVs have been shown to play key roles in various cellular processes as regulation, intercellular signalling, but also in infectious diseases where they facilitate spreading, and escape from immune surveillance [START_REF] Paolini | Biophysical properties of extracellular vesicles in diagnostics[END_REF][START_REF] Giannessi | The role of extracellular vesicles as allies of HIV, HCV and SARS viruses[END_REF][START_REF] Leclaire | A review of the biomechanical properties of single extracellular vesicles[END_REF] . So far, few methods are able to distinguish viruses from EVs [START_REF] Hoen | Extracellular vesicles and viruses: Are they close relatives[END_REF] . EVs used in this study were obtained from HIV or MLV producing cells and from uninfected cells. By using lipid bilayer markers DiO or DiL (Molecular Probes) we were able to fluorescently label EVs and to distinguish them from non enveloped viruses. All particles were depicted in Fig. 1A, and more precision can be found in Supporting Information.

Both particles, with existing fluorescent label and without initial fluorescent label, can be used. As for particles without initial fluorescent label, we used a one-step labelling procedure, which simply involved incubating the sample with a small volume of fluorophores (< 1µL) for a few minutes (10 to 15 min of incubation). By using several types of fluorophores, we were able to label a wide range of viruses as well as extracellular vesicles. Those fluorophores were passive markers, without interaction with the nanopores and therefore they did not alter the translocation of the viral particles (more information can be found in Supporting Information, S2). As mentionned, the particles were fluorescently labelled and then injected in the cis chamber of our setup. As shown in Fig. 1B, hydrodynamical driving induced by a pressure difference imposed by a microcontroller (MFCS, Fluigent) between the two sides of a nanoporous membrane (coated with a 50 nm thick gold layer on the trans side) was used to force translocation of particles from cis to trans side. The membranes used were track-etched membranes (Whatman, GE Polycarbonate) produced through heavy ion irradiation and they are commercially available dense arrays of cylindrical nanopores of controlled diameter and high density (1 × 10 8 pores/cm 2 ). After crossing the membrane, successful translocation events were detected by a Zero-Mode Waveguide (ZMW) setup. Briefly, the nanoporous array was illuminated by a laser beam (λ exc = 473nm) on the trans side and the gold layer inhibited the propagation of light through the membrane. It also induced an enhancement of the electromagnetical field in the nanopore on a depth equivalent to the radius of the pore whereas the electrical field was null at the surface of the membrane [START_REF] Levene | Zero-mode waveguides for single-molecule analysis at high concentrations[END_REF]40 . This effect enabled to optically separate the fluorescence signal of translocating particles from particles in the bulk (see Supporting Information and Ref [START_REF] Auger | Zero-mode waveguide detection of flow-driven DNA translocation through nanopores[END_REF] ). The main advantage of this method is its ability to detect events with a large parallelization (in our case 10 5 pores in parallel).

All types of particles used in this study were successfully detected at the exit of nanopores with a signal to noise ratio superior to 2. An example of average fluorescence intensity pattern for AAV particle can be found in Fig. 1C, as well as snapshots of particle exit. The fluorescence intensity was fitted by an exponential decay function and event durations were found to be 26±4 ms for AAV, which allowed us to confirm that we were in a ZMW regime.

Complementary experiences highlighting the benefits and relevance of the ZMW, as well as a simplistic ZMW translocation model, are available in Supporting Information (S2).

Thereafter, in order to be able to determine virus concentration with our ZMW setup, we first made a calibration of nanoporous membranes using fluorescent carboxyl polystyrene nanobeads (NPs, Spherotech Inc) from two batches (with sizes determined by DLS mea-surement) and of known concentrations. Nanobeads were smaller than pore size and acted as flow markers. Indeed, we assumed that the system was dominated by advection (Peclet number, P e > 1, see Supporting Information) and that the flow in our system verified the Hagen-Poiseuille law [START_REF] Kavokine | Fluids at the Nanoscale: From Continuum to Subcontinuum Transport[END_REF] . This was supported by the fact that we observed a linear increase of translocation frequency of nanobeads with pressure (see Supporting Information).

To perform calibration, we analysed the variation of nanobeads translocation frequency per pore, f , as a function of the concentration, C. The data for both types of nanobeads through pores of 400 nm and at applied pressure of 0.5 mbar were plotted on Fig. 2. The mean of the experimental slope for both types of NPs, α N P (9.7 × 10 -12 ± 0.7 × 10 -12 mL.particles -1 .s -1 ) was calculated and gave a direct relation between translocation frequency and concentration. We then compared α N P with a coefficient obtained theoretically to validate our calibration (see Supporting Information, S4).

Based on this calibration, we were then able to determine the viral particles concentration in biological samples. To do so, we measured the translocation frequency for series of dilutions originating from the same sample. The particle translocation frequency (f ) normalized by the particle translocation frequency for the initial sample (f 0 ) was represented as a function of the dilution ratio (r) on Fig. 3. f 0 was obtained by linear extrapolation from particle translocation frequency (f ) when r = 1. Each point corresponded to an experiment carried out with a single diluted sample and for each viral type. For each particle type, there were at least four different initial samples of unknown concentration. The linearity of the curve threw light on the fact that each particle going through a nanopore was effectively detected and no jamming effect occurred in the pore.

Initial concentration can then be obtained using calibration coefficient α N P and the translocation frequency for the initial sample (f 0 ) relying on the following formula: used in this study. Translocation frequency (f ) normalized by initial translocation frequency (f 0 ) as a function of dilution factor (r). For each particle type, different diluted solutions were made from the same initial sample. Pore diameter 400 nm, Applied pressure 0,5 mbar. Experimental errors were the standard error of the mean, and N replica > 24 for each experimental series.

C ZM W = f 0 α N P , (1)
We compared initial concentrations obtained for each biological sample to expected concentrations, that had been obtained by qPCR (for AAV and HBV samples) and NTA (for HIV, MLV and EVs samples). Results were presented in Fig. 4. We observed a good agreement between measured values using ZMW setup and expected values over six decades of concentrations. The slope of linear fit was equal to 0.96, which stood for 4% of error on our measurement. These results threw light on the versatility of our system, since we obtained a good precision for the concentration of all types of particles that were tested . It has to be recalled that each analysis was performed for 60 seconds, providing a total analysis duration of less than 15 minutes. Indeed, the total experimental duration included the incubation time required for the fluorescent labelling of the particles. However, incubation time can be performed at the beginning of an experiment set (for several samples) which allowed to save time.

Finally, given the large range of examined concentrations, we were then interested in determining the limit of detection of our system. To do so, we first determined the LOD theoretically by suggesting that it was reached when a single event was detected in our sytem, at given experimental conditions. As mentioned earlier, we assumed that the Hagen-Poiseuille law was valid. In this case, the flow through a single pore J, at applied pressure ∆P , can be expressed as follows: J = ∆P R h with R h = 8ηL πR 4 , R and L, respectively the radius and the length of the pore, η, the water viscosity.

Therefore, the total number of particles, N tot , at a given concentration C, going through the area of the observed surface S, during an acquisition time t, came as:

N tot = ∆P.N A .t.σ.S R h .C,
with σ the pore density and N A the Avogadro number. This led to the following formula for the limit of detection (LOD), which was defined as

0.5 1 1.5 2 2.5 3
C expected (particles/mL)

x 10 12 0.5 the concentration when the number of observed particles was equal to 1:

LOD = R h ∆P.N A .t.σ.S (2)
The LOD was then tested experimentally as shown on Fig. 5 on low concentration samples. We plotted the expected concentrations of different viral samples (HIV and AAV), C, as a function of the acquisition time, t. In order to prepare virus solutions at a given low concentration, a range of dilution was performed on a previously measured sample. The black dashed line corresponded to the theoretical LOD. Above this value, the violet circles represented the concentrations that could have been determined using our setup while the blue diamonds were the concentrations that couldn't be measured (no event detected). Almost all of the determined concentrations were above the theoretical LOD. Therefore, it represented a good estimation of the limit of our system. We concluded that the LOD of our system was about 10 5 particles/mL for an acquisition time of 60 seconds. Nevertheless, it was also possible to increase the acquisition time over 60 seconds to reach a lower LOD (for example about 1 hour to reach a LOD of 10 3 particles/mL).

Last but not least, since there is an ongoing need for direct counting of biotic nanoparticles such as viruses to probe the state of river water, we performed experiments on water coming from a river. Water was collected in the Rhône at Lyon (France, 4543 ′ 34.497 ′′ North, 449 ′ 14.402 ′′ East) on September 1st, 2021. We first filtered the sample through 0.45 µm pores. We then divided our sample into two parts and fluorescently labeled one of them with YOYO-1 and the other one with DiO. The concentrations obtained using the ZMW setup, were respectively 2.10 7 particles/mL for the YOYO-1 labelled sample and 4.10 7 particles/mL for the DiO labelled sample (more information can be found in Supporting Information, S3).

Labelling with YOYO-1 allowed us to detect and quantify viruses without envelope such as phytoplankton double-stranded DNA viruses (Myovirus, Podovirus, Siphovirus) [START_REF] Roose-Amsaleg | Utilization of interferometric light microscopy for the rapid analysis of virus abundance in a river[END_REF] or even The violet circle represented the concentrations that could have been determined using our setup while the blue diamonds showed the concentrations that couldn't have been measured (no event of translocation detected). Each point corresponded to a diluted sample originating from a viral particle sample whose concentration was determined previously.

Adenovirus and Norovirus that can be found in river water [START_REF] Sedji | Quantification of human adenovirus and norovirus in river water in the north-east of France[END_REF] . Whereas, labelling with DiO allowed us to detect and quantify both viruses with envelope (such as Herpesvirus, HIV, and Coronavirus including SARS-Cov2) [START_REF] Ye | Partitioning, and Recovery of Enveloped Viruses in Untreated Municipal Wastewater[END_REF][START_REF] Bibby | Identification of viral pathogen diversity in sewage sludge by metagenome analysis[END_REF][START_REF] Guerrero-Latorre | SARS-CoV-2 in river water: Implications in low sanitation countries[END_REF] and extracellular vesicles [START_REF] Roose-Amsaleg | Utilization of interferometric light microscopy for the rapid analysis of virus abundance in a river[END_REF] . The concentrations that was found with our method agreed with the values given in literature for other rivers such as the Marne (in Paris France) [START_REF] Roose-Amsaleg | Utilization of interferometric light microscopy for the rapid analysis of virus abundance in a river[END_REF] .

Conclusion

All together these results showed that a combination of nearfield optics technique and fluorescence provided a powerful tool to detect the translocation of individual virus through solid-state nanopores and to quantify their concentration.

Compared to other available methods, the main advantages of our strategy are that we achieved a low LOD and required a small volume of sample while keeping a short experimental duration and a specificity level that can be tuned by the final user. A comparison of the specifications and limits for different quantification methods can be found in the Supporting Information (S4).

More precisely, our technique allowed to work in real time, at the scale of the single particle and to consider the virus as a whole compared to other methods which quantify DNA or specific proteins. The system offered a good precision (4% of error on the concentration) and a low LOD which was adjustable according to the experimental parameters. Moreover, it was possible to quantify the viral particles directly from a complex biological samples (cell medium) comparable to clinical samples but also from real world samples (for example river water), while requiring small volumes. For viral samples whose concentrations were closed to the LOD (10 5 particles/mL), the required volume drop down to 20 µL. It must be added that for highly concentrated samples, a dilution step was required to ensure that the linear regime is observed. This can be easily achieved by making a range of dilutions from the same initial sample and by checking that the translocation frequency evolved linearly with the concentration. Working in a diluted regime was valuable to avoid interactions between viruses. We have added a complementary experiment in Supporting Information (S3), to provide additional information about the necessity of working in a diluted mode.

Besides, a major benefit of our system was its versatility since the fluorescent labelling can be adapted to a large range of virus type. By choosing specific fluorophores of different emission lengths it was also possible to detect a mixture of viruses in the same sample by matching the fluorescent tags to the sought viruses. Therefore, two viruses labeled with a different fluorophore could be distinguished. Besides, the use of specific fluorescent labels, prevented us to have signals coming from non-target particles.

This system also had the advantage of using low cost solid-state nanopores, since the nanoporous membranes used were track-etched polycarbonate membranes used for water filtration that allowed to detect events with a large parallelization (10 5 pores in parallel).

Last but not least, a miniaturization of our system can definitely be considered in the near future. The scope of applications of such a system could be numerous in basic research but also in clinic, for titration purposes.

Figure 1 :

 1 Figure 1: A. Characteristics of particles and experimental protocol used in this study. From top to bottom: VLP HIV, Virus Like Particle of Human Immunodeficience Virus; MLV, Murine Leukemia Virus; HBV, Hepatite B Virus; AAV, Adeno Associated Virus (serotype 8 and 9); EVs, Extracellular Vesicles. Fluorescent labelling of particles was required: it can be achieved by genome modification (GFP labelling for HIV and MLV) or directly by adding fluorophores in the sample (YOYO-1 for AAV and HBV, DiO for EVs). Potential cellular DNA is represented in red in VLP HIV and EVs, viral RNA in pink in MLV, and viral DNA in purple in HBV and AAV. Samples were then diluted. Sizes were determined by NTA for HIV, MLV and EVs and by Cryo-EM reconstruction for AAV 37 and HBV 38 . B. Zero Mode Waveguide setup for particles translocation through nanopores.The cis chamber contained the fluorescently labelled particles. Upon pressure application, the particles were pushed through the pore in the trans chamber and illuminated as soon as they cross the evanescent field region at the end of the pore. Once they have left the pore, they were unfocused and bleached.C. Evolution of fluorescence of event as a function of time and snapshots of particle exit. Normalized intensity was represented as a function of time for AAV (violet and red points, averaged on N=50 events). Normalized intensity was obtained by divided intensity by the maximum intensity. Time was rescaled to zero at the beginning of an event and red points corresponded to intensity before the event. Exponential decay was represented in blue. Pore diameter 400 nm, Applied pressure 0.5 mbar. Frame rate: 112 fps. Inset: image size=10 µm.

Figure 2 :Figure 3 :

 23 Figure 2: Calibration of the experimental setup using fluorescent polystyrene nanobeads (NPs). Translocation frequency (f ), as a function of NPs concentration (C), for two batches of NPs. Continuous blue and violet curves corresponded to linear fit. Pore diameter 400 nm, Applied pressure 0,5. Experimental errors were the standard error of the mean, and N replica > 36 for each experimental series.

Figure 4 :

 4 Figure 4: Comparison between concentrations obtained with our experimental setup (C ZM W ) and expected concentrations (C expected ) obtained with other methods (qPCR and NTA). Each point corresponded to a different sample. Results were depicted in log-log scale and also linear scale in the inset. Errorbars took into account standard error for f 0 and the error on the calibration coefficient α N P .

Figure 5 :

 5 Figure5: Expected concentrations (C) as a function of acquisition time (t). The black dashed line corresponded to the theoretical LOD. The violet circle represented the concentrations that could have been determined using our setup while the blue diamonds showed the concentrations that couldn't have been measured (no event of translocation detected). Each point corresponded to a diluted sample originating from a viral particle sample whose concentration was determined previously.

Acknowledgement

The authors thank Cendrine Moskalenko and Martin Castelnovo for their fruitful comments and discussions. This work was supported by the Centre National de la Recherche Scientifique under the 80 Prime project "NanoViro".

Supporting Information


PhD Sylvain Raynal
email: sylvain.raynal@univ-poitiers.fr

Ludovic Chatellier
email: ludovic.chatellier@univ-poitiers.fr

Professor Laurent David
email: laurent.david@univ-poitiers.fr

Axe Hydee

Dominique Courret
email: dominique.courret@imft.fr

Michel Larinier
email: michel.larinier@wanadoo.fr

Numerical simulations of fish-friendly angled trashracks at model and real scale

Keywords: Fish-friendly trashracks, Head loss, Numerical Modeling, RANS, Velocity distributions

. Real scale racks were then computed in order to evaluate the influence of both the bar spacing and the channel width on velocity distributions in real dimensions. The mesh generation and the numerical simulations were performed by the open source CFD software suite OpenFOAM. The trashrack solid comprised basic elements, individually created using CAD software and directly inserted in OpenFOAM's mesh generation utility. For small scale configurations, grid refinement was applied at the flume walls, at trashrack bars and downstream of the trashrack, whereas only bars were refined for real scale racks. The resulting number of cells ranged between 100,000 and 1,500,000. Steady state results were obtained by solving the Reynolds-averaged Navier-Stokes (RANS) equations for an incompressible and monophasic flow. The k-ε, k-ε-based RNG, k-ω, k-ω-based shear stress transport (SST) and Spalart-Allmaras models were examined to select the most appropriate one in terms of computation time and result accuracy. Results show that the k-ε-RNG is the model best agreeing with experimental results. Two-dimensional calculations seem to provide quite satisfactory results although both the head losses and the size of the recirculation zone downstream of the trashrack are slightly under-estimated. Real scale results confirm experimental ones and show that the bar spacing slightly effects upstream velocity profiles. Downstream of the rack, simulations with different flume width demonstrated that the size of the recirculation zone is proportional to the channel width.

INTRODUCTION

Fish mortality caused by turbines at hydropower plants during their downstream migration has become a worldwide concern in the last decade. In Europe, the European Water Framework Directive (2000/60/EC), the European Council regulation (no. 1100/2007) for the recovery of eel (Anguilla Anguilla) and restoration plans for amphibiotic species such as salmon (Salmo salar) or sea trout (Salmo trutta), have focused on the need to avoid or at least minimize these mortalities. Among the few answers, fish-friendly screens remain one of the main feasible. Indeed, conventional trash racks can be adapted into fish-friendly ones by reducing the bar spacing and either by inclining the rack from the ground, or by angling the rack from the wall. In both cases, the aim is to guide fish towards bypasses, located at the downstream end of the rack, by generating tangential currents.

Studying these racks requires studying both the biological aspects (guidance along the rack, possible impingement) and the economical ones (energy loss through the rack, homogeneity at the turbine entrance). Several numerical and/or experimental studies focused on some of these aspects in the case of angled trashracks. [START_REF] Ghamry | A numerical investigation to select a turbulence-closure model for simulating turbulent flows near trashracks (bar racks)[END_REF] numerically modeled free-surface water flowing through a rack. They compared their results with experimental ones, obtained by PIV (Particle Image Velocimetry) by Tsikata et al. (2007). These experiments mainly focused on velocities in the vicinity of the rack. Their racks comprised from 3 to 5 bars and had a slight angulation with α ranging from 90° (perpendicular rack) to 78°. Ghamry and Katopodis used the ANSYS CFX-11.0 CFD application to model a 3-D free surface turbulent flow using a 1 million cell mesh. They compared the performance of seven turbulence models (standard k-ε, standard k-ω ...). Five criteria including water levels, velocities and computational time, were selected to compare numerical and experimental results. On the whole, head losses were rather well predicted as well as velocities in the vicinity of the rack. Downstream of bars, velocity predictions differ somewhat from PIV measurements. The comparison with experiments revealed that the k-ω, k-ε and eddy viscosity transport models produce the best prediction. Finally, the k-ω was selected for other investigations since it showed slightly faster computational time.

Other studies are centered on the flow behavior around trashracks, without focusing on modeling the flow in the vicinity of the rack. Indeed, trashracks or fish screens may be modeled as porous media in order to lower the number of cells. [START_REF] Khan | Computational fluid dynamics modeling of turbine intake hydraulics at a hydropower plant[END_REF] numerically modeled the entrance of a hydropower plant (Star-CD software, high Re k-ε turbulence-closure model) and simplified the rack with this technique. Ho et al. ( 2011) also used a similar numerical modeling and replaced angled wedge-wire screens and perforated plates by porous media with directional porosity (Flow3D software, k-ε turbulence-closure model). Even with these simplifications, both studies resulted in numerical results in good agreement with experimental observations. [START_REF] Chatellier | Experimental characterization of the flow across fish-friendly angled trashrack models[END_REF] and [START_REF] Raynal | An experimental study in fish-friendly trashracks -Part 2. Angled trashracks[END_REF] experimentally investigated model angled racks with bars scaled down to half size. Different racks angles α (α = 30°, 45°, 60° or 90°) and different bar spacings e (e/b = 1, 1.5, 2 or 3 where b is the bar thickness) were tested in order to assess the effect of these parameters on both head losses and velocity distributions. They especially implemented ADV (Acoustic Doppler Velocimetry) and PIV systems in order to measure velocities around these racks. Results revealed that the upstream flow accelerated towards the end of the trashrack. At α =45°, the axial velocity U increased up to 70% above the upstream mean velocity V 1 . Downstream of the rack, the flow was asymmetric with a fairly large recirculation zone. The bar spacing and the bar shape had only a slight effect on upstream velocity profiles. For rectangular shaped bars, differences were up to 20% in the downstream part of the rack.

The present study focuses on calculations with angled racks, at both model and real scale. Experimental results from [START_REF] Chatellier | Experimental characterization of the flow across fish-friendly angled trashrack models[END_REF] and [START_REF] Raynal | An experimental study in fish-friendly trashracks -Part 2. Angled trashracks[END_REF] studies are used to validate computational results. Simulations with different turbulence models at model scale are performed to select the parameters producing the best agreement with experiments. Real scale simulations are then done in order to analyse the effect of the channel size or of the bar spacing on the velocity distribution upstream and downstream of the rack.

The second section of this paper explains the numerical parameters and describes the experimental results used for verification. The third section compares the efficiency of various turbulence-closure models in terms of head loss and velocity distribution predictions. Then, the best turbulence model is used in real scale simulation to analyze the influence of the bar spacing and the channel width's one on velocity profiles. All these results are then discussed and recommendations are made for the design of fish-friendly water intakes with angled trashracks.

NUMERICAL MODELING

This study focuses on two trashrack scales. First, computational results with model scale racks are compared with previous experimental results in order to validate the use of 2D turbulent flow with OpenFOAM in the context of this study, i.e. fish-friendly angled trashracks. Then, real scale racks are simulated to complete experimental results.

Experimental configurations selected for verification

Experimental results are extracted from [START_REF] Chatellier | Experimental characterization of the flow across fish-friendly angled trashrack models[END_REF] and [START_REF] Raynal | An experimental study in fish-friendly trashracks -Part 2. Angled trashracks[END_REF] studies. They performed an experimental investigation on model angled trashracks, with various rack angles (from α = 90 to 30°), bar spacings (from e/b= 1 to 3) and 2 bar shapes. Bars were half-scaled and were 5 mm wide by 40 mm deep. In addition, trashracks also comprised spacer lines and two lateral pieces, triangular shaped, used to hold the rack to the channel. The rack was inserted in a 0.6 m (B) wide open water channel, in which water flowed at Q = 130 l/s. Water depths were generally set at 350 mm leading to upstream mean velocities V 1 around 0.7 m/s. During this study, head losses were measured and velocity profiles were acquired at mid-depth with an Acoustic Doppler Velocimeter probe. These values are added to those measured on similar configurations with a PIV system by [START_REF] Chatellier | Experimental characterization of the flow across fish-friendly angled trashrack models[END_REF].

For the present study, two configurations, meeting fish-friendly criteria, have been selected for computations. Simulated racks are angled at α = 45°, comprise bars spaced by 10 mm (corresponding to 20 mm in real size), and have either a rectangular (PR) or a more hydrodynamic cross-section (PHillustrated in Figure 1). Head loss values and velocity profiles, measured in these two configurations, are detailed in section 3.2.

Mesh and numerical geometries

Model scale racks

Since model scale simulations are compared with experimental ones, model scale rack geometries need to be very close to those used during experiments. Simulated model racks comprised two specific lateral pieces and 51 bars, 5 mm thick (b) and 40 mm deep (p), spaced by e = 10 mm (Figure 1). Since simulations were two-dimensional, spacer lines could not be modeled. The rack, whose upstream end is placed at the origin of the coordinate system, is inserted in a 0.6 m wide domain which starts at x = -3 m and ends at x = 4m (the rack ends at x = 0.6 m).

Figure 1 Full-width (top) and zoomed (bottom) view of computed mesh for small scale racks, superimposed with resulting velocity fields (inlet velocity is uniformly set at 0.7 m/s). Bars are profiled (PH) with a rounded leading edge and a thin trailing edge. Velocity profiles, extracted for comparison at 20 mm from the rack (black dashed line) and downstream of the rack at x = 1 m (white dashed line), are also illustrated. Black arrows represent the coordinate origin.

Real size racks and water intakes

Real size racks also comprise bars and lateral pieces. Only rectangular bars are simulated. Bars are real scaled and are consequently 10 mm wide and 80 mm deep. The rack was also inserted at the origin of the coordinate system. Moreover, in all the real size configurations, the domain started at x = -B and ended at x = 3B.

The first kind of simulations focused on the effect of the bar spacing e on velocity profiles. The channel width was set at B = 10 m and the bar spacing was alternatively set at e = 10, 20 and 30 mm, leading to e/b ratios from 1 to 3.

The second type of computations was centered on the effect of the channel width on velocities. The bar spacing was fixed at e = 20 mm, and the channel width was alternatively set at B = 5, 10 and 30 m.

Mesh generation

Channel

The mesh was generated with the same OpenFOAM functionalities for both model and real scale simulations. The channel geometry is initially modeled as a coarse, structured, uniform rectangular mesh comprised of hexahedral cells. The chosen cell size represents the coarsest level of the mesh, also denominated as level "0". Incrementing the mesh level amounts to halving all the cell dimensions.

Trashrack

The trashrack geometry was generated in two phases. First, basic elements were created with CAD software. Then, according to selected parameters (channel width, bar spacing, bar width,…), the trashrack elements are scaled, rotated, and assembled using a C++ code.

Afterwards, this rack was inserted and meshed by the OpenFOAM's mesh castellation utility snappyHexMesh. Two complementary functions were used for mesh refinement: the mesh level around the inserted geometry, i.e. the trashrack, was specifically fixed and sub-domains, in which the mesh level was incremented, were created. All the mesh levels and increments refer to the level "0" defined during the channel geometry definition, and result in refined hexahedral and semi-hexahedral cells.

Mesh levels

Mesh levels were always the same for PR and PH racks. Table 1 compares some geometry and mesh parameters and also gathers refinement levels for both scales.

For real scale racks, the large number of bars prevented from having a very refined mesh and only bars were refined. Near bars, the size of the cell was around 1.5 mm.

For model scale racks, which comprise fewer bars, other zones were refined. Bars were refined with a cell size around 0.6 mm. In addition, longitudinal bands were refined near the channel sides in order to have good near-wall treatment. Typically, the band width was around B/20 (30 mm). Moreover, mesh refinement was also applied in the trashrack wake, over the whole channel width and until x = 2B.

Table 1

Mesh and geometry descriptions for small and real scale configurations Trashrack wake 2 0 * For real scale configurations, these values depend on the channel width B

Governing equations

Simulations were performed with OpenFOAM software, and using the "simpleFoam" solver, dedicated to stationary, turbulent and monophasic flows. The three dimensional (restricted to two dimensions using a single mesh layer the z-direction) Reynolds-averaged Navier-Stokes (RANS) equations were solved and were alternatively coupled with five turbulence-closure models:

-the k-ε developed by [START_REF] Launder | The numerical computation of turbulent flows[END_REF] -the k-ε based RNG method (renormalization group) by [START_REF] Yakhot | Development of turbulence models for shear flows by a double expansion technique[END_REF] -the k-ω by [START_REF] Wilcox | Multiscale model for turbulent flows[END_REF] -the k-ω based SST (shear stress transport) by [START_REF] Menter | Two-equation eddy-viscosity turbulence models for engineering applications[END_REF] -the [START_REF] Spalart | A one-equation turbulence model for aerodynamic flows[END_REF] model

Apart from the last one, all these turbulence models are two equation ones. Governing equations or detailed descriptions of these models can be found in original papers or in other studies such as [START_REF] Pope | Turbulent flows[END_REF] or ASCE task committee (1988).

Boundary conditions and initials values

Boundary conditions were the same for both model and real scale simulations. A symmetry condition is applied on top and bottom faces, leading to vertical velocities W = 0 m/s in the whole domain. At the inlet, a uniform velocity value is set at U = 0.7 m/s (corresponding to V 1 during experiments). Since only relative pressures are calculated by this solver, the pressure inlet value was set at 0. Bars and flume sides are considered as walls, with no-slip conditions. Specific near-wall functions, already implemented in OpenFOAM, were applied around these faces. At the outlet, zero gradient boundary conditions were applied on all variables.

Moreover, for each variable of the different turbulence models (k, ε, ω…), different initial values were tested. However, they had no significant effect on the final results and are not detailed herein.

SMALL SCALE COMPARISONS

Procedure

Model scale simulations aimed at selecting the best turbulence model for this study. Two main criteria were selected: computational time and agreement between experimental and numerical results. This second point is centered on three salient results:

-The head loss generated by the rack. In single phase numerical tests, head losses account for pressure differences only. Pressure values are extracted at x-positions similar to those where water levels were experimentally measured (upstream and downstream pressures are extracted at x = -1 m and x = 2.5 m respectively).

-The velocity distribution along the rack. Velocities are extracted at 20 mm from the rack in both numerical tests (black line in Figure 1) and PIV images.

-A transverse profile downstream of the rack highlighting the recirculation zone. As PIV images do not provide data further than x = 1 m, transverse velocity profiles are extracted at x = 1 m in numerical tests (white line in Figure 1).

Results

Upstream and downstream velocity profiles are compared in Figure 2 in order to assess the performance of each turbulence-closure model.

For velocity profiles along PR-racks, all turbulence models produce good predictions, reproducing the flow acceleration towards the end of the trashrack. Only slight differences occur between these models. Along PH-racks, velocities are over estimated with all turbulence models in the downstream half of the rack. One can hardly determine which model results in the best predictions of upstream velocity profiles. Indeed, only slight differences occur between upstream velocity profiles and there is no model resulting in good predictions for both bar shapes Downstream of each rack, the three dimensional behavior of the flow observed experimentally increases the differences between numerical predictions and measured velocities. Experiments show a recirculation zone extending on the left bank (y = 600 mm) until around y = 300 mm. The size of this recirculation is under estimated by all the turbulence models and for both bar shapes. Moreover, the k-ω, k-ω-SST and Spalart-Allmaras models seem to predict a velocity peak near the recirculation zone (around y = 0.37 m) which was not observed in measured velocity profiles. Nonetheless, downstream of PH-bars, the k-ε-RNG model produces the best estimation of the width of the recirculation zone. Head loss values and computational times, which are the other comparative criteria, are gathered in Table 2. In both configurations, head losses are under-estimated by numerical results. This can be explained by 3D patterns which cannot be modeled in such 2D simulations. Moreover, spacer lines, which block a fraction of the water depth, were not taken into account in these computations. Nevertheless, despite these numerical simplifications, some models result in good head loss predictions with errors lesser than 10%. The k-ω and k-ε-RNG models are those best predicting head losses for PR and PH bars respectively. On the contrary, the Spalart-Allmaras model produces the most under-estimated values.

However, if the k-ω and the k-ε-RNG prediction accuracies are rather similar, computational times are very different. Indeed, the k-ω model computational time, which was around five times as long as the k-ε-RNG one, finally appeared to be prohibitive.

Downstream of the rack -PH Downstream of the rack -PR Along the rack -PR Along the rack -PH

Table 2 Comparison of head loss coefficient predictions and computational time for small scale configurations. For each head loss numerical prediction, deviation from the experiments is written in brackets. The experimental uncertainty is also detailed for both bar shapes.

Turbulence models Head loss coefficient ξ

Computational time PR PH k-ε 2.8 (-15%) 2.19 (-9%) x 1.0 k-ε-RNG 2.83 (-14%) 2.24 (-7%) x 1.0 k-ω 3.02 (-8%) 2.2 (-8%) x 5.0 k-ω-SST 2.93 (-11%) 2 (-17%) x 5.0 Spalart-Allmaras 2.76 (-16%) 1.97 (-20%) x 1.3 Experiments 3.3 ± 6% 2.4 ± 8% --
In conclusion, all these five turbulence models result in relevant simulations. Nonetheless, results show that the k-ε-RNG model should be the best one for this study. Indeed, it is the fastest one and, on the whole, it best predicts velocity fields and head loss coefficients.

REAL SCALE SIMULATIONS

Extracted profiles

There are two main objectives in this fourth section focusing on real scale racks (Figure 3). First, simulations should help to determine the influence of the channel width B on velocity profiles, because B could not be changed during experiments. Secondly, simulations should also make it possible to determine the effect of the bar spacing on velocity profiles in real dimensions. This analysis is entirely achieved by centering on four velocity profiles extracted from computed results. Upstream velocities have been extracted along the rack, at 200 mm from bars. Downstream of the rack, three transverse velocity profiles have been extracted at x = 1.5B, 2B and 2.5B. Since the recirculation size may depend on the channel width, the axial position of downstream profiles was decided to be proportional to B. On the contrary, upstream of the rack, velocity profiles are more affected by the bar dimensions (which is the same in all configurations). Moreover, this order of magnitude (few decimeters) corresponds to recommended distances to describe velocity fields in front of screens with respect to fish guidance and impingement risks (USDI 2009, EA 2012).

Results

Figure 4 compares upstream and downstream velocity profiles for real scale trashracks with different bar spacings and different channel widths. These U/V 1 profiles are drawn against the coordinate y, normalized by the channel width B.

As observed experimentally, upstream velocity profiles are somewhat influenced by the bar spacing. When bars are closer, velocities tends to be lower in the downstream part of the rack. Velocity differences may range up to 10-15%. These differences tend to vanish downstream of the rack.

On the other hand, there is nearly no effect of the channel width on velocity profiles (drawn against y/B). This especially means that the width of the recirculation zone downstream of the trashrack is proportional to the channel width. For example, at x = 2B, axial velocities significantly decrease for all configurations for y/B > 0.65-0.70 and become negative for y/B > 0.85-0.90. Similar commentaries can be made for the length (along the x-direction) of this recirculation zone. Indeed, the good agreement between velocity profiles at x = 2B was also true for x = 1.5B and x = 2.5B (not shown in Figure 4), meaning that the size of the recirculation zone is proportional to the flume width B in both the x and y directions.

CONCLUSION

This study focused on two-dimensional numerical simulations of angled trashracks at both model and real scale. Two configurations angled at 45°, which were experimentally investigated by [START_REF] Chatellier | Experimental characterization of the flow across fish-friendly angled trashrack models[END_REF] and [START_REF] Raynal | An experimental study in fish-friendly trashracks -Part 2. Angled trashracks[END_REF], were selected for this study.

Numerical results at the same model scale were compared to these experimental results. Head losses predicted with simulations are slightly under-estimated. The two main reasons may be the absence of spacer lines or the two-dimensional simplification for these simulations. Nonetheless, the best turbulence closure models produce head loss estimations with more than 90% accuracy. Concerning computed velocities, upstream profiles are in good agreement with the experimental ones but, downstream of the rack, velocity fields are less satisfactory. Even if all the turbulence models generate a recirculation zone, computed velocity profiles are quite different from experimental ones. Indeed, the size of the recirculation zone is under-estimated and some turbulence models generate odd velocity features (velocity peaks, …).

On the whole, the five turbulence models finally resulted in rather similar results. Nevertheless, the k-ω and k-ε-RNG models were the best predicting ones. However, since simulation durations with the k-ε-RNG model were five times as fast as those with the k-ω one, the k-ε-RNG model was selected for large scale simulations. This remains coherent with other studies which also choosed k-ε based models (Ho et al. 2011[START_REF] Khan | Computational fluid dynamics modeling of turbine intake hydraulics at a hydropower plant[END_REF]. However, some studies like Gharmy and Katopodis (2009), who investigated trashrack configurations with rather low blockage ratio, identified the k-ω model as the best predicting one.

Still, in most studies focusing on trashracks, accurate numerical predictions require two equation turbulence closure models.

In summary, these model scale simulations validate the use of two dimensional computations to simulate fish-friendly angled trashracks. Although computed results somewhat differ from experimental ones, the main features are simulated and computational results remain relevant.

Real scale computations confirm some experimental results observed in small scale configurations. Bar spacing may influence the upstream velocity distribution along the rack. More especially, velocities are mainly different in the downstream end of the rack. Differences between e/b = 1 and e/b = 3 may range up to 20%. However, these differences tend to decrease downstream of the rack. Concerning the effect of the channel width, these computations also demonstrated that both upstream and downstream velocity profiles, drawn against y/B, are not affected by the channel width B. In particular, it was showed that the size of the recirculation zone is proportional to B.

All these conclusions, arising from this numerical study, complete previous experimental results on angled trashracks. They especially provide information at real scale and help to better understand the flow behavior downstream of the rack where highly asymmetric flows are generated. This recirculation zone, which can be penalizing for hydraulic operators, must be taken into account during water intake design phases.

Figure 2

 2 Figure 2 Velocity profiles along (top) and downstream of the rack (bottom) angled at α = 45°. Bar section is either rectangular (PR -left) or more hydrodynamic (PH -right). Velocities are normalized by the upstream mean velocity V 1 .

Figure 3

 3 Figure 3 Whole domain for real scale computations with B = 10 m. Velocity fields are superimposed with streamlines (inlet velocity is 0.7 m/s). The x-axis is represented at the bottom of the domain. Downstream velocity profiles are extracted at x = 15, 20 and 25 m.

Figure 4

 4 Figure 4 Velocity profiles along (left) and downstream of the rack at x = 2B (right). On the vertical axis, the transverse coordinate y is normalized by the channel width B. Horizontally, axial velocities are normalized by the upstream mean velocity V 1 .

ACKNOWLEDGEMENT

This work was funded by the European Regional Development Fund (ERDF), the Région Poitou-Charentes, ONEMA, ADEME, CNR, SHEM and EDF. Their support is greatly appreciated.


C Lasbrugnas

P Thomas
email: pthomas@unilim.fr

O Masson

J C Champarnaud-Mesjard

E Fargin

V Rodriguez

Second harmonic generation in poled tungsten tellurite glasses

Second harmonic signals have been successfully generated in thermally poled 85%TeO 2 -15%WO 3 glasses. Thermal poling of these glasses was undertaken at various temperatures and heating times using a voltage of 4•5 kV. The sample thickness was 500 µm. Second harmonic generation (SHG) in the poled glasses was analysed using the Maker fringes technique. After optimisation of the poling conditions (T=280°C, t=1 h), high c [START_REF] Dutreilh-Colas | New TeO2 based glasses for nonlinear optical applications: study of the Tl 2 O-TeO 2 -Bi 2 O 3 , Tl 2 O-TeO 2 -PbO and Tl 2 O-TeO 2 -Ga 2 O 3 systems[END_REF] values (about 1•6 pm/V) were obtained. It was demonstrated that this thermal poling treatment has generated optically nonlinear zones on the anodic surface. The thickness of the surface layer given a SHG was thin (5•22 µm), as classically observed with tellurite glasses. The possible reorientation, under electric field, of the glass structural entities could be the origin of such second order nonlinear properties.

Increasing interest in materials with nonlinear optical properties has been induced by the important industrial request for optical and electro-optical devices. Glasses are very good potential candidates for such applications because of their formability and their chemical stability. More particularly, tellurium dioxide based glasses are of special interest as their compositions can be readily adjusted due to their large glassy domains; they have low melting temperatures, good visible and infrared light transmittance, weak absorption coefficients and especially high linear (n 0 ) and nonlinear (n 2 , c 3 ) indices. [START_REF] El-Mallawany | Tellurite Glasses Handbook: Properties and Data[END_REF] The stereochemical activity of Te(IV) atoms is certainly at the origin of such properties. It is very often reinforced when either a second lone pair holder (Tl + , Bi 3+ …) or a d 0 cation (Nb 5+ , W 6+ …) is associated with the Te(IV) atoms. [START_REF] El-Mallawany | Tellurite Glasses Handbook: Properties and Data[END_REF][START_REF] Dutreilh-Colas | New TeO2 based glasses for nonlinear optical applications: study of the Tl 2 O-TeO 2 -Bi 2 O 3 , Tl 2 O-TeO 2 -PbO and Tl 2 O-TeO 2 -Ga 2 O 3 systems[END_REF][START_REF] Jeansannetas | Glass structure and optical nonlineraties in thallium(I) tellurium(IV) oxide glasses[END_REF] Nonlinear optics involves the study of phenomena induced by a modification of optical properties of a dielectric material under an intense beam. Basically, the nonlinear optical properties of transparent materials result from the generation of a polarisation P when an intense electromagnetic wave enters into these materials under an electric field E. This polarisation P can be expressed as a power series in the electric field E P=P 0 +c [START_REF] El-Mallawany | Tellurite Glasses Handbook: Properties and Data[END_REF] E+c [START_REF] Dutreilh-Colas | New TeO2 based glasses for nonlinear optical applications: study of the Tl 2 O-TeO 2 -Bi 2 O 3 , Tl 2 O-TeO 2 -PbO and Tl 2 O-TeO 2 -Ga 2 O 3 systems[END_REF] EE+c [START_REF] Jeansannetas | Glass structure and optical nonlineraties in thallium(I) tellurium(IV) oxide glasses[END_REF] EEE+… where P 0 is the permanent polarisation, c [START_REF] El-Mallawany | Tellurite Glasses Handbook: Properties and Data[END_REF] is the linear susceptibility which accounts for the linear optical index. c [START_REF] Dutreilh-Colas | New TeO2 based glasses for nonlinear optical applications: study of the Tl 2 O-TeO 2 -Bi 2 O 3 , Tl 2 O-TeO 2 -PbO and Tl 2 O-TeO 2 -Ga 2 O 3 systems[END_REF] and c [START_REF] Jeansannetas | Glass structure and optical nonlineraties in thallium(I) tellurium(IV) oxide glasses[END_REF] correspond, respectively, to the second and third order nonlinear susceptibilities. For a high intensity oscillating field E with frequency w, the second term is responsible for the generation of a double frequency 2w oscillating field, called second harmonic generation (SHG). This can appear only in non-centrosymmetric materials in which the second order nonlinear susceptibility c [START_REF] Dutreilh-Colas | New TeO2 based glasses for nonlinear optical applications: study of the Tl 2 O-TeO 2 -Bi 2 O 3 , Tl 2 O-TeO 2 -PbO and Tl 2 O-TeO 2 -Ga 2 O 3 systems[END_REF] is different to zero. Amorphous materials, such as glass, with an inversion symmetry at the macroscopic scale do not in principle generate second order optical nonlinearities. Nevertheless, it is now well recognised that is possible to break the inversion symmetry at a microscopic scale through a technique such as thermal poling and thus to induce second order nonlinearity. Thermal poling consists in applying a dc electric field at an elevated temperature (below the transition glass temperature T g ) and cooling the glass before removing the voltage. [START_REF] Myers | Large second-order nonlinearity in poled fused silica[END_REF] The mechanism underlying the formation of the nonlinearity in bulk glass is not yet completely understood. According to Myers et al, two different mechanisms can account for the induced second order nonlinear susceptibility: charge migration or dipole reorientation.

The model based on these two mechanisms can be expressed as [START_REF] Myers | Large second-order nonlinearity in poled fused silica[END_REF][START_REF] Mukherjee | Dynamics of secondharmonic generation in fused silica[END_REF] c [START_REF] Dutreilh-Colas | New TeO2 based glasses for nonlinear optical applications: study of the Tl 2 O-TeO 2 -Bi 2 O 3 , Tl 2 O-TeO 2 -PbO and Tl 2 O-TeO 2 -Ga 2 O 3 systems[END_REF] μc [START_REF] Jeansannetas | Glass structure and optical nonlineraties in thallium(I) tellurium(IV) oxide glasses[END_REF]

E dc +(Npb * /5kT)E loc
The first term is related to the interaction between the residual electric field E dc inside the material after removing the applied dc field and the third order nonlinearity. The second term is the resulting macroscopic second order nonlinearity induced by reorientation of polar bonds during poling treatment, each of them with a permanent dipole moment p (N is the number of permanent dipoles) and an hyperpolarisability b * , subject to a local field E loc .

Over the last ten years, it has been demonstrated that poling can induce second order nonlinearity in various tellurite glasses such as TeO 2 -ZnO, [START_REF] Tanaka | Second harmonic generation in electrically poled TeO2-based glasses[END_REF][START_REF] Shimizugawa | The relationship between glass structure and poling-induced optical second harmonic intensity for ZnO-TeO2 glasses[END_REF][START_REF] Narazaki | Induction and relaxation of optical second-order nonlinearity in tellurite glasses[END_REF] TeO 2 -WO 3 , [START_REF] Tanaka | Second harmonic generation in electrically poled TeO2-based glasses[END_REF][START_REF] Tanaka | Large optical second-order nonlinearity of poled WO3-TeO2 glass[END_REF] TeO 2 -B 2 O 3 , [START_REF] Tanaka | Optical second harmonic generation in poled MgO-ZnO-TeO 2 and B 2 O 3 -TeO 2 glasses[END_REF] TeO 2 -Nb 2 O 5 , [START_REF] Tanaka | Poling-induced structural change and second-order nonlinearity of Na+-doped Nb 2 O 5 -TeO2 glass[END_REF][START_REF] Yonesaki | Relaxation phenomena in second-order nonlinearity of thermally and optically poled Nb2O5 glasses[END_REF] TeO 2 -Nb 2 O 5 -Li 2 O, [START_REF] Tanaka | Second harmonic generation in electrically poled Li 2 O-Nb 2 O 5 -TeO 2 glasses[END_REF] TeO 2 -Na 2 O-Li 2 O, [START_REF] Narazaki | Effect of poling temperature on optical second harmonic intensity of lithium sodium tellurite glass[END_REF] TeO 2 -ZnO-MgO, [START_REF] Tanaka | Optical second harmonic generation in poled MgO-ZnO-TeO 2 and B 2 O 3 -TeO 2 glasses[END_REF][START_REF] Tanaka | Poling temperature dependance of optical second harmonic intensity of MgO-ZnO-TeO 2 glasses[END_REF] TeO 2 -ZnO-Na 2 O [START_REF] Narazaki | Effect of poling temperature on optical second harmonic intensity of sodium zinc tellurite glasses[END_REF] and TeO 2 -Pb(PO 3 ) 2 -Sb 2 O 3 . [START_REF] Ferreira | Second harmonic generation in poled tellurite glass[END_REF] The second order nonlinear susceptibilities, c [START_REF] Dutreilh-Colas | New TeO2 based glasses for nonlinear optical applications: study of the Tl 2 O-TeO 2 -Bi 2 O 3 , Tl 2 O-TeO 2 -PbO and Tl 2 O-TeO 2 -Ga 2 O 3 systems[END_REF] , measured were generally in the range 0•1 pm/V (TeO 2 -ZnO-MgO glasses) [START_REF] Tanaka | Optical second harmonic generation in poled MgO-ZnO-TeO 2 and B 2 O 3 -TeO 2 glasses[END_REF] -0•9 pm/V (TeO 2 -ZnO glasses), [START_REF] Narazaki | Induction and relaxation of optical second-order nonlinearity in tellurite glasses[END_REF] except for 80TeO 2 -20WO 3 (mol%) glass for which a very high c [START_REF] Dutreilh-Colas | New TeO2 based glasses for nonlinear optical applications: study of the Tl 2 O-TeO 2 -Bi 2 O 3 , Tl 2 O-TeO 2 -PbO and Tl 2 O-TeO 2 -Ga 2 O 3 systems[END_REF] of 2•1 pm/V was obtained. [START_REF] Tanaka | Large optical second-order nonlinearity of poled WO3-TeO2 glass[END_REF] Up to now, the most reliable models to explain the origin of SHG in tellurite glasses are based on either the migration of charged impurities, [START_REF] Narazaki | Induction and relaxation of optical second-order nonlinearity in tellurite glasses[END_REF][START_REF] Tanaka | Poling-induced structural change and second-order nonlinearity of Na+-doped Nb 2 O 5 -TeO2 glass[END_REF][START_REF] Yonesaki | Relaxation phenomena in second-order nonlinearity of thermally and optically poled Nb2O5 glasses[END_REF][START_REF] Narazaki | Infrared and XPS studies on the surface structure of poled ZnO-TeO2 glasses with second-order nonlinearity[END_REF] e.g Na + , towards the cathode side glass of the poled material or the possible reorientation, [START_REF] Tanaka | Second harmonic generation in electrically poled TeO2-based glasses[END_REF][START_REF] Shimizugawa | The relationship between glass structure and poling-induced optical second harmonic intensity for ZnO-TeO2 glasses[END_REF][START_REF] Tanaka | Optical second harmonic generation in poled MgO-ZnO-TeO 2 and B 2 O 3 -TeO 2 glasses[END_REF][START_REF] Tanaka | Poling temperature dependance of optical second harmonic intensity of MgO-ZnO-TeO 2 glasses[END_REF][START_REF] Narazaki | Effect of poling temperature on optical second harmonic intensity of sodium zinc tellurite glasses[END_REF] under the electric field, of the asymmetrical (TeO n ) structural units (where n varies from n=3 or 4 depending on the composition of glasses) in glassy samples with electric dipoles. This article aims to report SHG investigations, using thermal poling, on a 85TeO 2 -15WO 3 (mol%) compositional glass. The choice of this composition takes the following points into account: it has a good thermal stability, a high mechanical strength [START_REF] Blanchandin | Etude cristallochimique de quelques phases cristallisées et vitreuses appartenant aux systèmes TeO2-WO3 et TeO2-Nb2O5-Bi 2 O 3[END_REF] and an expectedly high third order nonlinear susceptibility c [START_REF] Jeansannetas | Glass structure and optical nonlineraties in thallium(I) tellurium(IV) oxide glasses[END_REF] . Moreover, this composition can be crystallised on heating to give the non-centrosymmetric metastable g-TeO 2 phase which exhibits SHG. [START_REF] Blanchandin | New investigations within the TeO2-WO3 system: phase equilibrium diagram and glass crystallisation[END_REF][START_REF] Champarnaud-Mesjard | Crystal structure, Raman spectrum and lattice dynamics of a new metastable form of tellurium dioxide: g-TeO2[END_REF] Studies are now in progress to control the crystallisation of g-TeO 2 phase nanoparticles in the bulk 85TeO 2 -15WO 3 (mol%) glass to promote SHG. These results on glass ceramics will be reported in a future paper. The present study details an investigation into SHG in 85TeO 2 -15WO 3 (mol%) glass, via an optimisation of the thermal poling conditions. The structural properties of the poled and unpoled glasses were studied using Raman spectroscopy. X-ray microprobe analyses were performed on both surfaces of the poled glasses.

Experimental

Glass preparation 85TeO 2 -15WO 3 (mol%) glasses were obtained using appropriate quantities of reagent grade TeO 2 and WO 3 (commercial product Interchim, +99%) as starting materials. TeO 2 was prepared by the decomposition at 550°C of commercial H 6 TeO 6 (Aldrich 99•9%). A small amount of Sb 2 O 3 (2 mol%) was added in order to lighten the glasses. Glassy pellets were prepared by melting the powder mixtures in platinum crucibles, at 800°C for 30 min. The melts were then rapidly quenched by flattening between two preheated brass blocks separated by a brass ring. This produced cylindrical samples 10 mm in diameter and 1•5 mm thick. The glasses were annealed at 300°C for 10 h (about 40°C below their glass transition temperature of 344°C) to remove the thermal stresses induced in the as quenched glasses. The samples were then mechanically polished to give 500 µm thick planar disks with parallel faces suitable for optical measurements. The resultant glasses were pale yellow and transparent as expected. Their homogeneity was checked using optical microscopy.

Thermal poling

The glass disks were thermally poled by using a home made apparatus. The 500 µm thick glass was sandwiched between two thin commercial borosilicate plates and kept in contact with stainless steel electrodes equipped with a heating stage. This equipment was placed inside a vacuum chamber. Different poling conditions were investigated. The glassy samples were heated up to temperatures ranging from 250°C to 310°C under a vacuum of about 10 -5 torr. After stabilisation of the temperature, a dc poling voltage was applied under different conditions (voltage from 3-4•5 kV, poling time from 20 min to 8 h). Then the glasses were cooled slowly to room temperature whilst maintaining the voltage and the vacuum. The voltage was switched off once the samples reached the room temperature.

Optical measurements

The optical transmission spectra of the unpoled and poled glasses were recorded at room temperature using a double beam spectrophotometer (AFAS-Monaco) in the wavelength range 400-1000 nm.

The second harmonic generation intensity of the thermally poled glasses was measured using the Maker fringes method. This method consists of varying the angle between the incident laser beam, with frequency w, and the glass sample containing at least one nonlinear optical planar layer. The intensity of the transmitted and generated 2w optical signal was then recorded by changing the anle in a periodic fashion. Simulations of the Maker fringes spectrum enables estimation of the second order nonlinear coefficient d 33 and the thickness of the nonlinear zone. It is worth noting that the nonlinear coefficient d 33 is directly linked to the second order susceptibility c [START_REF] Dutreilh-Colas | New TeO2 based glasses for nonlinear optical applications: study of the Tl 2 O-TeO 2 -Bi 2 O 3 , Tl 2 O-TeO 2 -PbO and Tl 2 O-TeO 2 -Ga 2 O 3 systems[END_REF] by d 33 =½c [START_REF] Dutreilh-Colas | New TeO2 based glasses for nonlinear optical applications: study of the Tl 2 O-TeO 2 -Bi 2 O 3 , Tl 2 O-TeO 2 -PbO and Tl 2 O-TeO 2 -Ga 2 O 3 systems[END_REF] . [START_REF] Boyd | Nonlinear Optics[END_REF] The SHG Maker fringes were analysed using a new general ellipsometrical analysis for planar multilayered systems. [START_REF] Rodriguez | General Maker-fringe ellipsometric analyses in multilayer nonlinear and linear anisotropic optical media[END_REF] The whole Maker fringe signal is recorded for two polarisation configurations: pp and sp polarisation, where pp and sp refer to the linearly polarised incident beam with a polarisation parallel to the plane of incidence (pp) or with a polarisation perpendicular to the plane of incidence (sp).

The set up for SHG measurements is shown in Figure 1. It was composed of a Q-switched Nd/YAG laser operating at a wavelength of 1064 nm with a 30 Hz repetition rate and a 10 ns pulse width. A beam attenuator placed just after the laser source enabled adjustment of the pulse energy at the sample. The po-larised source beam was split into two rays by a beam splitter: one ray was used to record the fundamental (w) intensity using a near infrared diode detector; the other, which was firstly polarised by a glan polariser, passed through a half wave (1064 nm) rotation plate and a set of appropriate filters and then was focused on the sample with a spot of 10 µm diameter. The harmonic (2w) transmitted signal was collected by a detection unit consisting of a 1064 nm selective reflector, a bandpass filter (532 nm), a rotation analyser and finally a photomultiplier tube. The reflected fundamental or harmonic wave was simultaneously collected by a second detection unit with a double goniometer q-2q coupling. Because data may contain errors due to fluctuations between laser pulses, the collected intensities were averaged over 100 pulses. Absolute calibration of the SHG intensities was obtained using z-cut quartz and taking c 11 =0•6 pm/V as a reference. [START_REF] Dmitriev | Handbook of nonlinear optical crystal[END_REF] This setup also has enabled estimation of the linear refractive index at w and 2w by collecting the 2q reflected wave around the Brewster angle over the [15-80°] q range. A LiNbO 3 crystal in phase matching condition was used as a 532 nm source to record the reflected signal. It is very important to determine two linear refractive indices at 1064 and 532 nm because these values indicate the dispersion in the indices and provide a very good indication of the coherence length of the material.

Surface characterisation

Structural studies of the poled glasses were undertaken using Raman scattering. The Raman spectra were recorded in the 100-1200 cm -1 range using a Dilor spectrometer (XY model), equipped with a CDD detector and an Ar + laser (514•5 nm excitation line) in a backscattered geometry. The diameter of the laser spot focused on the sample was about 1 µm. Measurements were made at low power (<100 mW) of the excitation line, in order to avoid any damage to the samples. The spectral resolution was about 4•5 cm -1 at the excitation line.

Quantitative compositional analysis was undertaken using an X-microprobe in EDS (energy dispersion spectroscopy) mode on both surfaces of the poled glass. Measurements were made with a 40 nA electron beam and an accelerating voltage of 20 kV, corresponding to a sodium detection limit of 340 ppm (0•0340 wt% or 0•085 at%). The diameter of the electron beam was 10 µm.

Results and discussion

Ultraviolet-visible absorption and linear refraction indices

The optical transmittance of the glass is an important parameter to determine before beginning simulation of the measured second harmonic signal because it gives information about the dispersion inherent in the material. The 85TeO 2 -15WO 3 (mol%) glass has a large domain of transparency which varies from 500-1000 nm (Figure 2(a)). Nevertheless, thermal poling blackens the surface on contact with the cathode which increases the ultraviolet-visible absorption of the glass due to reflection losses or 'lost' diffusion (Figure 2(b)). After a slight polishing of this face, the glass recovers its properties (Figure 2(c)) and the second order nonlinearity is conserved. Moreover, the maximum of transmission stabilised at 80%, is due to many reflections at the glass faces. This transmission maximum indicates that this glass should possess high linear refractive indices (n 0 >2) as classically observed with tellurite glasses. This is confirmed by measurement of these indices using the Brewster angle reflection method: n 0 =2•193(±0•001) for the wavelength 532 nm and n 0 =2•087(±0•001) for the wavelength 1064 nm.

Second harmonic generation

As SHG in 85%TeO 2 -15%WO 3 glasses has never been studied, different thermal poling conditions (temperature and time) have been investigated to optimise the second harmonic signal. Second order nonlinear coefficients d 33 deduced from Maker fringe simulations and the nonlinear zone depths are reported in Table 1 for different poling conditions. Significant second harmonic signals were observed for all poling conditions. The surface SH active layer (3-17 µm) was obtained at the anode side.

No coherence is observed between the different values of the d 33 coefficient and the depths of the nonlinear zone. These results are not evidence of real evolution with respect to the poling temperature or the poling time. Nevertheless, some main features can be underlined. The values of the d 33 coefficients are about 0•3-0•4 pm/V, which are high values compared to those of other tellurite glasses or oxide glasses. The best compromises lie in a poling temperature about 50°C lower 532 nm or 1064 nm filter, analyser and photomultiplier tube. The first detection unit PM1 enables the transmitted SHG signal to be detected, and a coupled rotation (2q) of the second detection unit PM2, with respect to the sample angle (q), enables reflection measurements to be undertaken than the glass transition temperature T g (344°C) and a poling time that is not too long, but is long enough to obtain an electric equilibrium under the electric field (1 or 2 h). For too high poling temperatures (>300°C) or too long poling times (>2 or 4 h with respect to the heating temperature), glasses are damaged by fracture and/or blackening. The highest value of the d 33 coefficient was obtained under the following optimised conditions of poling: 280°C for 1 h under a dc voltage of 4•5 kV. Figure 3 shows experimental and simulated Maker fringes for these poling conditions. The corresponding second order nonlinear susceptibility c 2 is 1•6 pm/V, one of the highest values ever observed in tellurite glasses. This value can be linked to the third order nonlinear susceptibility c 3 which is very high for this glass (c 3 ª300×10 -23 m 2 /V 2 , measured at l=0•8 µm). The SH active layer is very thin (L=5•22 µm).

The chemical compositions of an unpoled glass and of two glasses poled under extreme conditions (heating at 250°C for 8 h and heating at 295°C for 30 min), have been checked using an x-ray microprobe. Both surfaces of the poled glasses, that are in contact with electrodes during the poling process, have been analysed. The values reported in Table 2 are average ones and it is important to underline that the percentage of each element present has shown no variation along the anode or the cathode surfaces. These analyses clearly show that no noticeable modification of glass compo-sition occurs after poling. In the two poled samples, the appearance of a significant amount of sodium at the anode side can be observed. Sodium was only observed on the anode side (atomic percentage>detection limit of sodium) and not on the cathode side (the atomic percentage of Na could not be extracted from the measurement noise). Moreover a cross sectional analysis of these samples has not shown any Na + composition gradient from the anode side towards the cathode side. The sodium at the anode side, which is relatively low, is certainly due to diffusion from the borosilicate plate through the face in contact with the anode during the poling treatment. These results indicate that, no Na + cations migrate from the anodic side towards the cathodic side, contrary to what has been observed in other tellurite systems [START_REF] Narazaki | Induction and relaxation of optical second-order nonlinearity in tellurite glasses[END_REF][START_REF] Tanaka | Poling-induced structural change and second-order nonlinearity of Na+-doped Nb 2 O 5 -TeO2 glass[END_REF][START_REF] Yonesaki | Relaxation phenomena in second-order nonlinearity of thermally and optically poled Nb2O5 glasses[END_REF][START_REF] Narazaki | Infrared and XPS studies on the surface structure of poled ZnO-TeO2 glasses with second-order nonlinearity[END_REF] and under different poling conditions.

The Raman spectrum of the 85TeO 2 -15WO 3 (mol%) glasses is shown in Figure 4. This spectrum is quite similar to the Raman spectrum of pure TeO 2 glass. [START_REF] Sekiya | Structural study of WO3-TeO2 glasses[END_REF][START_REF] Shaltout | Structural studies of tungstate-tellurite glasses by Raman spectroscopy and differential scanning calorimetry[END_REF][START_REF] Noguera | Vibrational and structural properties of glass and crystalline phases of TeO2[END_REF] . It has two main bands situated near 450 and 650 cm -1 , which correspond respectively to the symmetric vibrations of the Te-O-Te bridges and to the asymmetric vibrations of the Te-O terminal bonds, both co-existing in TeO 2 glass. This indicates that the glassy network is mainly comprised of TeO 4 units. The band located near 930 cm -1 corresponds to W=O stretching vibrations. No significant differences can be observed between the spectrum obtained from an unpoled sample and the spectrum obtained from a poled sample (Figure 4). It is worth noting that the spectra recorded on both faces of the poled glass are identical. The only weak difference between spectra of the unpoled and poled glasses can be seen on the broad Table 1. Second order nonlinear coefficient (d 33 :d 33 = ½c [START_REF] Dutreilh-Colas | New TeO2 based glasses for nonlinear optical applications: study of the Tl 2 O-TeO 2 -Bi 2 O 3 , Tl 2 O-TeO 2 -PbO and Tl 2 O-TeO 2 -Ga 2 O 3 systems[END_REF] )) and SH layer thickness (L), for different thermal poling conditions, of glasses with 85TeO 2 -15WO 3 (mol%) composition (voltage: 4•5 kV and thickness of samples: 500 µm) band situated in the range 700-800 cm -1 , which is attributed to the asymmetric vibrations of the Te-O terminal bonds. The intensity of this band seems to have slightly increased after poling. This could mean that no significant structural modification is induced by thermal poling (TeO 4 units are conserved, no breaking of the tellurite glass network, only the Te-O terminal bonds are involved) and that only a reorientation of these glass structural TeO 4 entities occurs under electric field. The presence of a 5s 2 electronic lone pair on Te(IV) atoms certainly enhances this reorientation. This mechanism could be at the origin of the SHG in tungsten tellurite poled glasses.

Conclusion

85TeO 2 -15WO 3 (mol%) glasses have been poled and have shown a better SHG performance than silicate glasses (generally <1 pm/V) and most other tellurite glasses. After optimisation of the thermal poling conditions, a high second harmonic signal (c [START_REF] Dutreilh-Colas | New TeO2 based glasses for nonlinear optical applications: study of the Tl 2 O-TeO 2 -Bi 2 O 3 , Tl 2 O-TeO 2 -PbO and Tl 2 O-TeO 2 -Ga 2 O 3 systems[END_REF] =1•6 pm/V) was obtained under the following conditions: T=280°C, t=1 h, V=4•5 kV. The surface SH active layer was thin (5•22 µm) and occurred at the anode side. Both poled surfaces, in contact with electrodes during the poling treatment, were analysed by Raman spectroscopy and x-ray microprobe analysis. No migration of sodium towards the cathodic surface was found. No significant structural modification was observed, and only a reorientation of the TeO 4 glass structural entities can be the origin of SHG in 85TeO 2 -15WO 3 (mol%) glasses.

Figure 1 .

 1 Figure 1. Schematic diagram of the experimental setup for SHG acquisitions: BA: beam attenuator; BS: beam splitter; D: near infrared diode; F: 1064 nm filter or LiNbO 3 crystal and 532 nm filter; PM1: 532 nm filter, analyser and photomultiplier tube; PM2: 532 nm or 1064 nm filter, analyser and photomultiplier tube. The first detection unit PM1 enables the transmitted SHG signal to be detected, and a coupled rotation (2q) of the second detection unit PM2, with respect to the sample angle (q), enables reflection measurements to be undertaken

Figure 2 .

 2 Figure 2. Optical transmission spectra of the 85TeO 2 -15WO 3 (mol%) glass: (a) unpoled glass; (b) poled glass; (c) poled glass polished on cathode side

Figure 3 .

 3 Figure 3. Experimental (lines with circles) and simulated (continuous lines) Maker fringes for the 85TeO 2 -15WO 3 (mol%) glass poled at 280°C for 1 h under a voltage of 4•5 kV

Figure 4 .

 4 Figure 4. Raman spectra of the 85TeO 2 -15WO 3 (mol%) glass: (a) before poling; (b) after poling on anode side; (c) after poling on cathode side

Table 2 .

 2 Percentages of different atoms constituting the 85%TeO 2 -15%WO 3 poled glass (esd are given in parentheses)

	Atom	Before	Poling conditions
		poling	250°C, 8h	295°C, 30 min
			Anode Cathode	Anode Cathode
			(at%)	(at%)	(at%)	(at%)
	Te	27•2	27•0(2) 27•2(2)	27•1(2) 27•3(2)
	O	68•2	67•9(6) 68•2(6)	68•0(6) 68•2(6)
	W	4•6	4•5(2)	4•6(2)	4•5(2)	4•5(2)
	Na

Acknowledgement

C. Lasbrugnas would like to express her gratitude towards the European Community (European Social Fund) and the Limousin Region for their financial support of the present work.