William Bakhache

Aymeric Neyret

Joe Mckellar

Camille Clop

Eric Bernard

James Weger-Lucarelli

IRIM Laurence Briant
email: laurence.briant@irim.cnrs.fr

James Weger- Lucarelli

Fatty acid synthase and stearoyl-CoA desaturase-1 are conserved druggable cofactors of Old World Alphavirus genome replication

Keywords: Chikungunya virus, Mayaro virus, lipid metabolism, fatty acid synthase, stearoyl-CoA desaturase-1, genome replication CHIKV: Chikungunya virus, MAYV: Mayaro virus, FASN: fatty acid synthase, SCD1: stearoyl-CoA desaturase, PI3K: phosphatidylinositol 3-kinase, nsP3: non structural protein 3

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Introduction

Alphaviruses are widespread arthropod-borne viruses responsible for several medically important emerging diseases. Their impact on human health has been illustrated in the last decade by the rapid spread of the Chikungunya virus (CHIKV) that causes an acute transient febrile arthralgic illness, and may lead to chronic incapacitating articular disease [START_REF] Burt | Chikungunya virus: an update on the biology and pathogenesis of this emerging pathogen[END_REF]. At the beginning of the 21 st century, CHIKV reemerged in East Africa and spread to countries neighboring the Indian Ocean before invading India, South-Eastern Asia, Caribbean Islands and South America, with autochthonous transmission episodes also reported in Europe [START_REF] Pezzi | GloPID-R report on Chikungunya, O'nyong-nyong and Mayaro virus, part I: Biological diagnostics[END_REF]. Over the last 15 years, CHIKV has caused at least 5 million autochtonous cases in >40 countries. CHIKV belongs to the Semliki Forest virus sero-complex in Old World Alphaviruses. In this phylogenetic clade, CHIKV genome displays up to 40 % nucleotide divergence with Mayaro virus (MAYV) [START_REF] Lavergne | Mayaro virus: Complete nucleotide sequence and phylogenetic relationships with other alphaviruses[END_REF]. First isolated in 1954 in Trinidad and Tobago, MAYV generates a self-limiting acute viral disease that may evolve towards chronic arthralgia very similar to CHIKV [START_REF] Anderson | An epidemic exanthem associated with polyarthritis in the Murray valley[END_REF][START_REF] Causey | Mayaro virus: a new human disease agent. III. Investigation of an epidemic of acute febrile illness on the river Guama in Pará, Brazil, and isolation of Mayaro virus as causative agent[END_REF]. While responsible for sporadic and small outbreaks in south and central American countries around the Amazon basin [START_REF] Azevedo | Mayaro Fever Virus, Brazilian Amazon[END_REF][START_REF] Izurieta | Hunting in the Rainforest and Mayaro Virus Infection: An emerging Alphavirus in Ecuador[END_REF][START_REF] Leduc | An outbreak of Mayaro virus disease in Belterra, Brazil. II. Epidemiology[END_REF][START_REF] Neumayr | Mayaro Virus Infection in Traveler Returning from Amazon Basin, Northern Peru[END_REF][START_REF] Schmidt | Epidemic jungle fever among Okinawan colonists in the Bolivian rain forest. II. Isolation and characterization of Uruma virus, a newly recognized human pathogen[END_REF][START_REF] Talarmin | Mayaro virus fever in French Guiana: isolation, identification, and seroprevalence[END_REF], the recent detection of MAYV in Brazil and Haiti raises growing concerns about MAYV potential to spread to other regions similar to CHIKV in the past [START_REF] Lednicky | Mayaro Virus in Child with Acute Febrile Illness, Haiti, 2015[END_REF][START_REF] Mackay | Mayaro virus: a forest virus primed for a trip to the city?[END_REF]. Currently, there is no vaccine against any of these Alphaviruses and no specific FDA-licensed drug for routine use is available. The limited efficacy of therapies tested so far for the management of CHIKV disease, including ribavirin [START_REF] Ravichandran | Ribavirin therapy for Chikungunya arthritis[END_REF], the anti-malaria drug chloroquine [START_REF] Roques | Paradoxical Effect of Chloroquine Treatment in Enhancing Chikungunya Virus Infection[END_REF] and corticosteroids [START_REF] Javelle | Specific Management of Post-Chikungunya Rheumatic Disorders: A Retrospective Study of 159 Cases in Reunion Island from 2006-2012[END_REF] points at the urgent need for basic antiviral research to identify the first therapeutic targets and antiviral molecules effective against Alphaviruses.

Cell membranes are critical for almost all steps of (+)RNA virus life cycle. The exploitation of membranes for initial interactions of the virion with the host cell, penetration and fusion of the viral particle, or for assembly and budding of enveloped progeny virions has long been demonstrated [START_REF] Altan-Bonnet | Lipid Tales of Viral Replication and Transmission[END_REF]. More recently, a key role has been assigned to cell membranes during the replication phase of (+)RNA viruses. This step generally occurs in host membrane-derived replication organelles whose creation and appropriate composition is pivotal for genome replication (Z. Zhang et al., 2019). Remodeling the cell membranes to create autophagosomes, despite being reported as having either pro-or antiviral function according to the viral species, has also found critical for the replication of various viral species [START_REF] Jackson | Viruses and the autophagy pathway[END_REF]. Being the major components of membranes, lipids therefore play active roles in the virus life cycle. As such, lipids and associated metabolism are attractive targets for the development of antiviral therapeutics.

Fatty acids are basic building blocks for the majority of cellular lipids and are therefore a source of components necessary for increased membrane production. The core reaction of fatty acid synthesis is catalyzed by the fatty acid synthase (FASN), a homodimeric enzyme that condensates one molecule of acetyl-CoA with seven molecules of malonyl-CoA to generate the 16-carbon saturated fatty acid palmitate. Starting from free fatty acids, stearoyl-CoA desaturase (SCD1) also known as Δ-9desaturase, catalyzes the biosynthesis of monounsaturated fatty acids (MUFAs) used as precursors for the synthesis of various lipids including phospholipids, triglycerides and cholesteryl esters. Once incorporated in membranes, fatty acids, and its derivatives, depending on the length and saturation degree of their hydrocarbon tail, exert a structural effect on membrane fluidity, permeability, and flipflop dynamics among others and also alter the function of integral membrane proteins [START_REF] Bond | Fatty Acid Desaturation and Elongation in Mammals[END_REF]. Both FASN and SCD1 lipogenic enzymes are controlled by the sterol regulatory element-binding protein (SREBP) family members, the master transcriptional factors placed under control of the phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin (PI3K/AKT/mTOR) signaling axis [START_REF] Furuta | Fatty acid synthase gene is up-regulated by hypoxia via activation of Akt and sterol regulatory element binding protein-1[END_REF]. Recently, Alphaviruses were reported to manipulate the PI3K/AKT/mTOR signaling axis through the presence of conserved sequences in virus-encoded nonstructural protein 3 (nsP3) with consequences on their host nucleotide, amino acid and lipid metabolisms [START_REF] Mazzon | Alphavirus-induced hyperactivation of PI3K/AKT directs pro-viral metabolic changes[END_REF]. According to this information, this study was therefore conceived to explore the functional role of FASN and SCD1 during Old World Alphavirus infection and to explore their relevance as therapeutic targets for antiviral purpose. Using RNA interference and pharmacological drugs, we demonstrate that FASN and SCD1 expression and enzymatic activity are required for optimal CHIKV genome replication.

We demonstrate that their proviral function is conserved for MAYV, thereby reporting among the very first host co-factors for this poorly studied pathogen. Finally, we provide evidence that the US Food and Drug Administration (FDA)-approved anti-obesity drug Orlistat, an inhibitor of FASN activity is active against CHIKV and MAYV infection. Altogether, these data point at the intricate relationship existing between arthritogenic Old World Alphaviruses and fatty acid metabolism in their vertebrate host and highlight the therapeutic potential of targeting fatty acid biosynthesis and desaturation to limit the multiplication of a broad range of Alphaviruses in human cells.

Material and Methods

Cells

HEK293T cells (ATCC number ACS-4500) used for infection experiments and Vero cells (ATCC number CCL-81) used for titration of the CHIKV strains were cultured in Dulbecco's modified Eagle's medium (DMEM, Thermo Fischer Scientific) supplemented with penicillin and 10% fetal calf serum (FCS, Lonza) and grown at 37 °C in a 5% CO2 atmosphere. The C6/36 mosquito cell line, derived from A. albopictus, was grown at 28 °C and 5% CO2 in Minimal Essential Medium (MEM) (Sigma) complemented with 10% inactivated FCS and 1% antibiotics. Cell viability was measured using the Cell Titer 96 Aqueous one solution cell proliferation assay kit (Promega) according to the manufacturer's protocol.

Reagents and antibodies

Quercetin, cerulenin and Orlistat were purchased from Sigma Aldrich. CAY10566 was from Abcam.

Drugs were diluted in DMSO. Appropriate concentration of DMSO was used in control experiments.

Antibodies against FASN, SCD1 and GAPDH were purchased from Santa Cruz Biotechnologies, Inc. Anti-tubulin and anti-actin mAbs were from Sigma-Aldrich and Na + /K + ATPase mAbs were purchased from Abcam. Rabbit polyclonal serum against nsP1 was a generous gift from Pr. Andres Merits (Tartu University, Estonia). Alexa-647-conjugated wheat germ hemagglutinin (WGA) and secondary antibodies were from Thermo Fisher Scientific.

Viruses

The CHIKV reporter virus was produced from the pICRES1 full-length CHIKV genomic clone (LR2006_OPY1 strain) containing the renilla luciferase marker inserted into the region encoding nsP3 [START_REF] Pohjala | Inhibitors of alphavirus entry and replication identified with a stable Chikungunya replicon cell line and virus-based assays[END_REF]. The CHIKV-mCherry-377 was previously described [START_REF] Kummerer | Construction of an infectious Chikungunya virus cDNA clone and stable insertion of mCherry reporter genes at two different sites[END_REF].

CHIKV RNA was transcribed in vitro from the SP6 or T7 promoter using the mMESSAGE mMACHINE kit (Ambion-Life Technologies). RNA (1 μg) was then transfected with lipofectamine 2000 (Thermo Fisher Scientific) into 10 5 HEK293T cells. The MAYV reporter virus was obtained from an infectious clone derived from the complete sequence of the TRVL 4675 strain with a nano-luciferase coding sequence inserted in the nsP3 gene and cloned under the control of the CMV promoter [START_REF] Chuong | Infectious cDNA clones of two strains of Mayaro virus for studies on viral pathogenesis and vaccine development[END_REF].

The virus was produced by transfection of the infectious clone in Vero cells. Fourty-eight hrs after transfection, the supernatants were collected, filtered through a 0.45 µm membrane, and tittered using plaque assay as previously reported [START_REF] Bernard | Endocytosis of Chikungunya Virus into Mammalian Cells: Role of Clathrin and Early Endosomal Compartments[END_REF].

Plasmids and transfection

The nsP1 sequence was amplified by PCR using pCHIKV-LR-5'GFP (LR-OPY strain) as a template and cloned into the pEGFP-C1 plasmid as previously reported [START_REF] Matkovic | The host DHX9 DExH Box helicase is recruited to Chikungunya virus replication complexes for optimal genomic RNA translation[END_REF]. CHIKV reporter transreplication system (kindly provided by Andres Merits) was composed of the CMV-P1234 plasmid encoding a CMV-driven CHIKV nonstructural ORF and of the HSPolI-Fluc-Gluc pseudogenome template containing a Firefly luciferase and a Gaussia luciferase reporter genes under control of CHIKV genomic and subgenomic promoters respectively [START_REF] Utt | Design and use of Chikungunya virus replication templates utilizing mammalian and mosquito RNA polymerase I mediated transcription[END_REF]. Plasmids were transfected using JetPei reagent (Polyplus Transfection). Twenty-four hrs post-transfection, replication was monitored using the Dual-Glo luciferase assay kit (Promega) and a Spark luminometer (Tecan). Values were normalized according to protein content in the sample determined using the BCA Assay (Pierce).

RNA interference

siRNA targeting FASN or SCD1 mRNA were purchased from Integrated DNA technologies and Sigma-Aldrich, respectively. Non-targeting siRNA were from Dharmacon. Transfection was achieved using Interferin (Polyplus Transfection). At 48 hrs post-transfection, an aliquot of the cells was harvested to determine the silencing efficiency by Western blotting. The remaining cells were infected with CHIKV.

Cell fractionation and membrane flotation assays

Cells were incubated in hypotonic buffer (10mM Tris/HCl [pH 7.4], 10 mM NaCl supplemented with protease inhibitors) for 10 min on ice and then lysed with a Dounce homogenizer (30-40 strokes). The lysates were clarified by low-speed centrifugation at 1000 g for 10 min. Post-nuclear supernatants were then adjusted to a final concentration of 500 mM NaCl and incubated for 30 minutes on ice.

Cytosolic fraction (supernatant, S25) and membrane fraction (pellet, P25) were obtained by ultracentrifugation at 25,000 g for 20 minutes in an MLA-150 rotor (Beckman Coulter). P25 samples were solubilized in lysis buffer composed of 1% Brij 96 in 20mM Tris-Cl pH 7.5 before analysis.

Immunoblotting

Samples were separated by SDS-PAGE, then transferred to a PVDF membrane (Hybond, Amersham).

Membranes were blocked against nonspecific binding by using 5% non-fat milk in PBS, 0.1% Tween 20, and probed with appropriate primary antibodies. After washings in PBS containing 0.1% Tween 20, the membranes were incubated with HRP-conjugated secondary antibodies. After final washes, the detection was performed by incubating the membranes with Luminata Forte (Merck) and then image acquisition was made using a Chemidoc (Bio-Rad). Band intensity has been determined utilizing ImageJ software.

Immunofluorescence microscopy and image analysis

Cells grown on glass coverslips were washed with PBS and then fixed with 4% paraformaldehyde in PBS (Sigma Aldrich) for 10 minutes. Then, the cells were permeabilized with 0.1% Triton-X100 in PBS and blocked for 30 minutes with PBS containing 0.2% bovine serum albumin. Incubation with primary antibody was performed at 37°C for 1 hr at room temperature and secondary reagents were added for 30 minutes at 37°C. DAPI (Sigma-Aldrich) was used to stain the nucleus. Staining with WGA-Alexa 647 was performed by incubation at room temperature for 10 minutes. After final washes, coverslips were mounted with Prolong Gold antifade mounting media (Thermo Fisher Scientific). Images were acquired using a Leica SP5-SMD scanning confocal microscope equipped with a 63×, 1.4 numerical aperture Leica Apochromat oil lense at the Montpellier Resources Imaging platform.

Statistical analysis

All of the analyses (unpaired Student's t-test) were performed using GraphPad Prism version 6 (GraphPad Software Inc.). * p<0.01; ** p<0.001; ***p<0.0001; **** p<0.0001.

Results

PI3K activity is essential for CHIKV infection

The PI3K/AKT/mTOR signaling axis is essential for the control of cell proliferation, protein translation and anabolic activities within the cell. Under nutrient abundance, mTOR activation stimulates aerobic glycolysis, and de novo lipid synthesis especially through the control of FASN expression (Figure 1) [START_REF] Furuta | Fatty acid synthase gene is up-regulated by hypoxia via activation of Akt and sterol regulatory element binding protein-1[END_REF]. Starting from the recent observation that Ross River virus (RRV) and Semliki Forest virus (SFV), both grouped with Old World arthritogenic Alphaviruses, enhance glycolysis and biosynthesis of C16:0 (palmitate) and C18:0 (stearate) fatty acids in their host by activating the PI3K/AKT/mTOR pathway [START_REF] Mazzon | Alphavirus-induced hyperactivation of PI3K/AKT directs pro-viral metabolic changes[END_REF], we investigated the functional importance of this signaling axis in CHIKV infection. For this purpose, we took advantage of the plant flavonoid quercetin (3,3ʹ,4ʹ,5,7-pentahydroxyflavone), a well-characterized pharmacological PI3K inhibitor [START_REF] Agullo | Relationship between flavonoid structure and inhibition of phosphatidylinositol 3-kinase: A comparison with tyrosine kinase and protein kinase C inhibition[END_REF]. Quercetin, used in a concentration range that was controlled to have no toxic effect (Supplementary Figure 1A), was added to HEK293T cells 30 min before challenge with a CHIKV reporter virus encoding a luciferase gene inserted in nsP3 sequence (Figure 2A; upper panel) (multiplicity of infection (MOI) = 1). After 24 hrs in culture, the infection was monitored by quantification of luciferase expression in cell extracts. In these conditions, quercetin showed a concentration-dependent inhibitory effect on CHIKV infection (Figure 2A; lower panel). Host lipid metabolism is expected to be pivotal in the various steps of enveloped virus's life cycle. To discriminate between inhibition of viral entry/fusion or subsequent steps in the CHIKV replication cycle, we next used a CHIKV transreplication system (depicted in Fig 2B ; upper panel). Cells were transfected for 2 hrs with equal amounts of CMV-P1234 and HSPolI-Flu-Gluc plasmid to reproduce CHIKV post-entry replication [START_REF] Utt | Design and use of Chikungunya virus replication templates utilizing mammalian and mosquito RNA polymerase I mediated transcription[END_REF], before the addition of increasing concentrations of quercetin to the culture medium. In these conditions, quercetin displayed a dose-dependent inhibitory effect on virus-encoded luciferase activities (Figure 2 B, C), supporting that inhibition occurred at the genome replication step. These results were consistant with post-infection experiments performed using a replicating CHIKV (Supplementary Figure 2A).

Altogether, these results, therefore, designate the PI3K/AKT/mTOR signaling axis as essential for CHIKV post-entry events.

Fatty acid synthase regulates CHIKV genome replication

The PI3K/Akt/mTORC1 signaling has been previously reported to be molecularly connected with FASN expression [START_REF] Sande | Role of the Phosphatidylinositol 3ʹ-Kinase/PTEN/Akt Kinase Pathway in the Overexpression of Fatty Acid Synthase in LNCaP Prostate Cancer Cells[END_REF]. Indeed, in our hands, quercetin-mediated PI3K targeting was able to reduce FASN protein levels in HEK293T cells (Figure 2D). According to this information and to quercetin anti-CHIKV activity reported above, we explored the direct contribution of FASN in CHIKV genome replication. HEK293T cells were transfected with siRNA specific for FASN or with a non-targeting control duplex. After 48 hrs, FASN expression was monitored by immunoblotting, showing that FASN protein level was reduced by over 70% by targeting FASN siRNA (Figure 3A). Then, the cells were transfected with CMV-P1234 and HSPolI-Fluc-Gluc plasmids. In these conditions, reduction of FASN protein level correlated with a 3-fold reduced luciferase activities (Figure 3B), attesting that FASN expression is required for CHIKV genome replication. Mammalian FASN is a multifunctional enzyme with 7 different catalytical activities that catalyzes the reaction leading to the production of palmitate, a 16-carbon fatty acid. Activity of the N-terminal β-ketoacyl-synthase and C-terminal thio-esterase in FASN can be inhibited using cerulenin antibiotic ((2R)(3S)-2,3-epoxy-4-oxo-7,10-dodecadienolyamide) and the FDA-approved anti-obesity drug Orlistat (tetrahydrolipstatin), respectively (Schcolnik-Cabrera et al., 2018) (Figure 1). Using these drugs, and the CHIKV transreplication system, we next explored whether FASN enzymatic activities are required for genome replication. CHIKV-encoded luciferase activities were dramatically inhibited by cerulenin at concentrations above 15 µM (Figure 3) that were non-toxic (Supplementary Figure 1). A similar effect (>80% inhibition) was also observed in the presence of 50 or 100 µM Orlistat (Figure 3E,F). Despite reducing cell viability by around 20%, this dramatic effect clearly attests of Orlistat potent anti-CHIKV activity. Interestingly, both drugs were also effective at inhibiting CHIKV infection in C6/36 cells (Supplementary Figure 3). Altogether these results support that expression and enzymatic activity of FASN are required for optimal CHIKV genome replication. This result was further validated using CHIKV-infected cells treated with cerulenin and Orlistat after virus exposure (Supplementary Figure 2B,C).

Stearoyl-CoA desaturase 1 expression and enzymatic activity are required for CHIKV genome replication

Starting from saturated fatty acids, preferentially palmitoyl-CoA and stearoyl-CoA, the reaction product of FASN, SCD1 introduces a double bond between carbons 9 and 10, to yield palmitoleoyl-CoA and oleoyl-CoA, respectively. To evaluate the impact of fatty acid desaturation on the CHIKV viral cycle, SCD1 was knocked down using siRNA, leading to the almost complete inhibition of SCD1 expression (Figure 4A). When these cells were transfected with the CHIKV plasmids, expression of the luciferase reporter genes was dramatically reduced as compared to cells transfected with non-targeting control siRNAs (Figure 4B). SCD1 activity can be manipulated by CAY10566 (3-[4-(2-chloro-5-fluorophenoxy)-1-piperidinyl]-6-(5-methyl-1,3,4-oxadiazol-2-yl)-pyridazine), a potent inhibitor of fatty acyl-CoA conversion to monounsaturated long-chain fatty acids [START_REF] Liu | Discovery of Potent, Selective, Orally Bioavailable Stearoyl-CoA Desaturase 1 Inhibitors[END_REF]. Taking advantage of this property, we questioned the contribution of SCD1 enzymatic activity in CHIKV replication. In the presence of increasing concentrations of CAY10566, CHIKV-encoded luciferase actitivies were significantly decreased up to 50%, indicating that SCD1 activity is required for optimal CHIKV genome replication (Figure 5C). Altogether, these results identify SCD1 as an essential novel cofactor of CHIKV whose enzymatic activity is required for genome replication events and therefore demonstrate that desaturation of fatty acids is pivotal to post-entry events.

FASN and SCD1 are conserved cofactors of Old World Alphavirus replication

While reported at risk of spreading to new environments, the biology of MAYV is almost unknown and no antiviral molecule has been identified against this virus. We therefore investigated the inhibitory effect of lipid metabolism inhibitors on MAYV in vitro. Because this virus remains understudied, we first established that it actively replicates in human epithelial HEK293T cells (Supplementary Figure 4).

A parallel infection with a MAYV-Luc reporter virus (Figure 5A) and a CHIKV-Luc (MOI of 1) demonstrated that after 24 hrs in culture, they generated a comparable luciferase activity in the cell lysate. Using this cell model, MAYV infection was investigated in the presence of quercetin, cerulenin, Orlistat and CAY10566 to determine its requirement for PI3K, FASN and SCD1 activity respectively.

Inhibitors were added either 1.5 hr before (Figure 5 B-E) or 1.5 hr after the viral challenge (Figure 5 F-I). As previously observed for CHIKV, each inhibitor induced a dose-dependent inhibitory effect on MAYV-directed luciferase reporter gene expression independent of the fact that it was added before or after viral challenge. These results, therefore, parallel those obtained with CHIKV, indicating that PI3K, FASN and SCD1 have conserved proviral activity during genome replication of distantly related Old World Aphaviruses.

Outcome of FASN and SCD1 in Alphavirus-replicating cells

We next investigated whether FASN and SCD1 protein level are manipulated in Alphavirus-infected cells. Since antibodies are lacking for MAYV, we used CHIKV as a model for subsequent studies. First, FASN and SCD1 expression was analyzed overtime upon CHIKV infection using immunoblotting experiments. As shown in Figure 6A, FASN and SCD1 protein levels remained almost unchanged until 16 hrs of infection. At this time, a slight decrease was observed reflecting CHIKV toxicity. FASN was previously reported to be recruited to replication foci formed CHIKV-infected [START_REF] Karlas | A human genome-wide loss-of-function screen identifies effective chikungunya antiviral drugs[END_REF], a result that was confirmed by us (data not shown). We therefore investigated the consequences of CHIKV infection on SCD1 subcellular localization. In control cells, SCD1 staining accumulated in the cytoplasm (Figure 6B) with a distribution coherent with the previously reported endoplasmic reticulum localization of this protein [START_REF] Nguyen | Stearoyl Coenzyme A Desaturase 1 Is Associated with Hepatitis C Virus Replication Complex and Regulates Viral Replication[END_REF]. This pattern persisted in infected cells throughout the time of the infection experiment. No clear colocalization could be observed with the CHIKV nonstructural proteins (nsPs) taking part to the replication complex. According to these results, FASN and SCD1 behave differently upon CHIKV infection, with FASN possibly recruited to replication complexes as previously reported [START_REF] Karlas | A human genome-wide loss-of-function screen identifies effective chikungunya antiviral drugs[END_REF] while this could not be evidenced for SCD1. These differences may reflect difference in the proviral function of these enzymes in Alphavirus replication cycle.

Consequences of fatty acid metabolism manipulation on CHIKV nsP1 binding to host membranes

Alphavirus genome replication is ensured by the four nsPs complexed with the RNA genome in bulbshaped membranous organelles derived from the host plasma membrane [START_REF] Spuul | Assembly of alphavirus replication complexes from RNA and protein components in a novel trans-replication system in mammalian cells[END_REF]. In these compartments, the replication machinery is tightly anchored to the lipid bilayer thanks to nsP1, the viral methyl/guanylyltransferase. Membrane anchoring is mediated through a central a-helix (position 244-263 in the CHIKV protein) and reinforced by the palmitoylation of conserved cysteines at the Cterminus (position 417-419 in CHIKV nsP1) [START_REF] Peranen | The alphavirus replicase protein nsP1 is membrane-associated and has affinity to endocytic organelles[END_REF][START_REF] Utt | Design and use of Chikungunya virus replication templates utilizing mammalian and mosquito RNA polymerase I mediated transcription[END_REF]. Both nsP1 palmitoylation and the host-encoded ZDHHC2 and ZDHHC19 palmitoyl transferases involved in acylation are required for CHIKV infectivity [START_REF] Utt | Design and use of Chikungunya virus replication templates utilizing mammalian and mosquito RNA polymerase I mediated transcription[END_REF][START_REF] Zhang | Fatty Acid Synthase Promotes the Palmitoylation of Chikungunya Virus nsP1[END_REF]. Given the pivotal role of FASN in palmitate synthesis, we investigated the consequences of FASN inhibition on nsP1 membrane binding capacity. HeLa cells expressing a CHIKV GFP-nsP1 protein were left untreated or maintained in the presence of cerulenin or Orlistat at concentrations that were effective against CHIKV replication. In control cells, confocal microscopy imaging detected the GFP-nsP1 fluorescence colocalized with Alexa647-conjugated wheat germ hemagglutinin that stains the cell membranes (Figure 7A). Cell fractionation, clearly evidenced the presence of the GFP-nsP1 protein in the membranous (P25) fraction that also stained positive for the Na + /K + ATPase membrane-associated protein. NsP1 was in contrast detected at low level in the cytosolic (S25) fraction (Figure 7B). In the presence of of cerulenin, the GFP-nsP1 protein detected by confocal microscopy was more diffuse in the cytoplasm (Figure 7A). After cell fractionation, an increased proportion of this protein was also detected in the S25 sample and concomitantly slightly reduced in the P25 sample (Figure 7B). This phenotype was further exacerbated starting from cells cultured in the presence of Orlistat. Therefore, cerulenin-and Orlistat-mediated inhibition of FASN activity and the expected associated decrease in availability of fatty acids in the cell reduce nsP1 membrane association. According to these results, the reduced CHIKV genome replication generated by FASN inhibitors may partly reflect a decrease in nsP1 anchoring to membranes, a capacity required for optimal activity of CHIKV replication machinery as recently reported [START_REF] Utt | Design and use of Chikungunya virus replication templates utilizing mammalian and mosquito RNA polymerase I mediated transcription[END_REF].

Discussion

This study was conceived to question the interplay between CHIKV infection and the host fatty acid metabolism in human cells. Using RNA interference and pharmacological inhibitors, we show that FASN and SCD1 expression and activity are required for optimal CHIKV genome replication. FASN is pivotal for various RNA viruses including Respiratory Syncytial Virus [START_REF] Ohol | Direct Inhibition of Cellular Fatty Acid Synthase Impairs Replication of Respiratory Syncytial Virus and Other Respiratory Viruses[END_REF], West Nile Virus [START_REF] Martin-Acebes | West Nile virus replication requires fatty acid synthesis but is independent on phosphatidylinositol-4phosphate lipids[END_REF], Astrovirus [START_REF] Murillo | Identification of Host Cell Factors Associated with Astrovirus Replication in Caco-2 Cells[END_REF], hepatitis C virus (HCV) [START_REF] Tongluan | Involvement of fatty acid synthase in dengue virus infection[END_REF][START_REF] Yang | Fatty acid synthase is up-regulated during hepatitis C virus infection and regulates hepatitis C virus entry and production[END_REF] and dengue virus (DENV) [START_REF] Huang | Hepatitis C virus replication is modulated by the interaction of nonstructural protein NS5B and fatty acid synthase[END_REF][START_REF] Tongluan | Involvement of fatty acid synthase in dengue virus infection[END_REF]. Its enzymatic activity regulates important metabolic processes. First, palmitate, the byproduct of FASN is required for post-translational protein acylation. Here, we show that cerulenin and Orlistat, two well known FASN inhibitors that reduce palmitate availability, decrease nsP1 membrane binding capacity.

The role of nsP1 acylation in membrane anchoring of the Alphavirus replication complex has been long reported [START_REF] Laakkonen | The effects of palmitoylation on membrane association of Semliki forest virus RNA capping enzyme[END_REF]. While dispensable for Sindbis virus infectious cycle [START_REF] Ahola | Effects of Palmitoylation of Replicase Protein nsP1 on Alphavirus Infection[END_REF][START_REF] Zusinaite | Mutations at the palmitoylation site of non-structural protein nsP1 of Semliki Forest virus attenuate virus replication and cause accumulation of compensatory mutations[END_REF], the conserved nsP1 acylated cysteines have been recently found critical for CHIKV replication [START_REF] Utt | Design and use of Chikungunya virus replication templates utilizing mammalian and mosquito RNA polymerase I mediated transcription[END_REF]. Moreover, FASN inhibition by C75 was previously reported to reduce CHIKV nsP1 palmitoylation (N. Zhang et al., 2019). Accordingly, cerulenin and Orlistat anti-CHIKV activity reported herein may partly result from the decreased nsP1 membrane binding capacity.

Besides, it may also directly result from a reduced availability of FASN reaction products, palmitate (16:0) and stearate (18:0), for complex lipid biosynthesis. By catalyzing their conversion into palmitoleate (16:1n-7) and oleate (18:1n-9), SCD1 regulates the saturated fatty acids to MUFA balance with consequences for ATP production via b-oxidation and energy storage in the form of triglycerides.

It also regulates for the biosynthesis of phospholipid fatty-acyl chains, triglycerides and cholesterol esters pivotal for membrane extension, fluidity and curvature [START_REF] Ralston | SCD1 inhibition during 3T3-L1 adipocyte differentiation remodels triacylglycerol, diacylglycerol and phospholipid fatty acid composition[END_REF]. Here, by using siRNA and taking advantage of the inhibitor CAY10566, we demonstrate that SCD1 expression and desaturase activity are required for CHIKV post-entry events thereby identifying SCD1 as a new CHIKV proviral factor. SCD1 has already been identified as an important cofactor for the replication of Flaviviruses, namely DENV [START_REF] Hishiki | Stearoyl-CoA desaturase-1 is required for flavivirus RNA replication[END_REF], Zika virus and yellow fever virus [START_REF] Gullberg | Stearoly-CoA desaturase 1 differentiates early and advanced dengue virus infections and determines virus particle infectivity[END_REF] in mammalian cells. A screen performed to identify cellular factors and functions required for Brome mosaic virus (BMV) RNA replication in yeast also designated OLE1 the yeast homolog of SCD1 as a proviral factor for viral RNA synthesis. [START_REF] Lee | Mutation of host delta9 fatty acid desaturase inhibits brome mosaic virus RNA replication between template recognition and RNA synthesis[END_REF]. For HCV, genome replication defect observed upon SCD-1 blocade was directly correlated with an alteration virus-induced membranous replication organelles as a consequence of modifications in membrane composition [START_REF] Lyn | Stearoyl-CoA desaturase inhibition blocks formation of hepatitis C virus-induced specialized membranes[END_REF][START_REF] Nguyen | Stearoyl Coenzyme A Desaturase 1 Is Associated with Hepatitis C Virus Replication Complex and Regulates Viral Replication[END_REF]. The exact function of SCD1 and MUFA in CHIKV post-entry replication events remains to be determined. Nevertheless, requirement for a functional SCD1 suggests that cerulenin and Orlistat antiviral effect not only results from the above discussed reduced nsP1 membrane binding capacity, but may also a reflect a decreased fatty acids availability for MUFA biosynthesis. This result therefore prompts the investigation of MUFA contribution in CHIKV life cycle. Interestingly, we observed a similar requirement for an enzymatically active FASN and SCD1 for MAYV genome replication as found for CHIKV. Despite a generally good conservation of virus-host interactions among Old world Alphaviruses, some differences have been reported [START_REF] Thaa | Differential Phosphatidylinositol-3-Kinase-Akt-mTOR Activation by Semliki Forest and Chikungunya Viruses Is Dependent on nsP3 and Connected to Replication Complex Internalization[END_REF]. Our results provide among the very first information on an understudied Alphavirus with high risk of emergence and suggest the possibility to transpose some knowledge acquired from CHIKV.

Altogether these results provide evidence that Old World Alphavirus genome replication is tightly interconnected with host lipid metabolism. This observation may open novel avenues for the elaboration of antiviral strategies with broad spectrum application in this group of viruses. De novo lipogenesis is currently considered an emerging therapeutic target [START_REF] Escribá | Membrane-lipid therapy: A historical perspective of membrane-targeted therapies -From lipid bilayer structure to the pathophysiological regulation of cells[END_REF]. More specifically, FASN activity can be modulated through pharmacological drugs that are well tolerated in humans.

Mainly, Orlistat, a derivative of lipstatin, approved by the FDA [START_REF] Ballinger | Orlistat: its current status as an anti-obesity drug[END_REF], active against CHIKV and MAYV in our hands, displays antiviral activity against Coxsackievirus B3 [START_REF] Ammer | The anti-obesity drug orlistat reveals anti-viral activity[END_REF], HCV [START_REF] Nasheri | Modulation of fatty acid synthase enzyme activity and expression during hepatitis C virus replication[END_REF] and DENV [START_REF] Tongluan | Involvement of fatty acid synthase in dengue virus infection[END_REF]. The wide spectrum antiviral properties of this anti-obesity drug suggests its possible repurposing for anti-infectious applications.

The recent development of liver-targeted SCD1 inhibitors [START_REF] Lachance | Discovery of potent and liver-targeted stearoyl-CoA desaturase (SCD) inhibitors in a bispyrrolidine series[END_REF] and of the oral SCD1 inhibitor Aramchol, evaluated in patients with primary nonalcoholic fatty liver disease and nonalcoholic steatohepatitis [START_REF] Ajmera | MRI Assessment of Treatment Response in HIV-associated NAFLD: A Randomized Trial of a Stearoyl-Coenzyme-A-Desaturase-1 Inhibitor (ARRIVE Trial)[END_REF][START_REF] Safadi | The Fatty Acid-Bile Acid Conjugate Aramchol Reduces Liver Fat Content in Patients With Nonalcoholic Fatty Liver Disease[END_REF] further supports the possibility to target lipogenic enzymes as a promising avenue for the elaboration of antiviral strategy applicable to medically important Alphaviruses.

Figure 1 :

 1 Figure 1: Fatty acid biosynthesis pathway and inhibitory drugs used in this study.

Figure 2 :

 2 Figure 2: Pharmacological inhibition of the PI3K/Akt/mTOR/FASN axis and consequences on CHIKV

Figure 3 :

 3 Figure 3: FASN expression and enzymatic activity are required for CHIKV genome replication. (A)

Figure 4 :

 4 Figure 4: Consequences of SCD1 inhibition on CHIKV genome replication. (A) HEK293T cells

Figure 5 :

 5 Figure 5: FASN and SCD1 have proviral activity for MAYV replication. HEK293T cells were infected

Acknowledgments

This work was funded by the Agence Nationale de la Recherche (ANR-18-CE11-0026-01). WB is a fellow of Montpellier Méditerranée Infections -Infectiopôle Sud foundation. We are grateful to Pr Andres Merits (University of Tartu, Estonia) for providing us with the reporter CHIKV viruses, replicons and anti-nsP1 antibodies.

Luciferase activity was monitored after 24 hrs. Values were normalized according to protein content in the sample.


Mapping the globe. The patterns of mega-ships

Port Technology International (2018), No. 77, pp. 94-96 César DUCRUET, Justin BERLI Centre National de la Recherche Scientifique (CNRS) How is the shipping network of the largest containerships distributed across the globe? Many issues have been raised about the so-called mega-ships in terms of port capacity and competitiveness, shipping alliances, and technological change, but the precise geography of their circulation had remained somewhat overlooked to date. This article thus provides a concrete description and explanation of the observed pattern in the recent period, notwithstanding a brief explanation of the data and methodology used for such a purpose.

Data and methodology

The most efficient way to map and analyse the global container shipping network is to extract information from the Lloyd's List Intelligence database, produced by the world's leading maritime intelligence and insurance in history1 . Covering the complete months of June and December 2016, our database comprises 121 mega-ships defined by a capacity over 14,000 TEUs (i.e. 1.98% of the total number of containerships), with a total fleet capacity of 1,976,077 TEUs (i.e. 9.62% of the total containership capacity). Mapping the combined traffic of all containerships (including mega-ships) for 2016 consisted in assigning "real flows" to a world maritime grid at both nodes (ports) and links (segments)2 . Lloyd's information being vessel movements between ports without knowledge on their exact circulation pattern or spatial trajectory, such a method allowed for representing flows with accuracy following the principle of the shortest path. A port-to-port matrix was thus elaborated including not only direct movements between ports (i.e. A-B, B-C, C-D) but also indirect linkages (AC, BD) in order to consider the full voyage of the ships. As a preliminary result and among the 1,000 ports connected by containerships in 2016, only 67 have welcomed mega-ships, thus only 6.7% of world ports. This subgroup of the world container fleet is thus highly selective in its network design and port choice when designing its services and routes.

The global pattern of mega-ship traffic

The cartography of the distribution of global containership traffic first confirms the importance of the round-the-world trunk line connecting the main economic centres of the world or "Triade" in the Northern hemisphere, with smaller routes being more north-south, south-south, or intra-regional (Figure 1). What is absolutely striking is the fact that mega-ship traffic (illustrated in dark colour) remains spatially concentrated along the Europe-Asia route, without any presence elsewhere. A number of factors can explain such a network structure and specialisation. First of all, the maritime silk road has long been a major highway since Antiquity and prolonged in colonial times as well as nowadays with the now famous One Belt, One Road vision and strategy of China. Second, the expansion of the Panama Canal was not sufficient to welcome such sea giants, so that their circulation pattern remains bound to this particular segment of the global, East-West trunk line. Despite recent announcements in the press about the launch of mega-ships on the Transpacific route (Knowler, 2015), the long-term effects of the 2009 global financial crisis had the major effect to concentrate the services of alliances and individual shipping lines on the more lucrative Europe-Asia segment.

Implications for ports

A zoom on this specific Europe-Asia route allows figuring the port hierarchy along this route as well as the share of mega-ship traffic (%TEUs) in the total containership traffic of ports (Figure 2). Such results reveal that port size is not always reflected by the absolute or relative importance of mega-ship traffic (see also Table 1). Statistically speaking, the linear (Pearson) correlation coefficient between total traffic and mega-ship traffic is only about 0.44, while the power-law line based on a log-log fit of the same variables reached only 0.38 (R²). The direct implication of these results is that other factors than size come to play, mainly related to the location and function of ports.

As a matter of fact, ports with a lower share of mega-ship traffic are often gateway ports which main function is to serve hinterlands. However, this same share is also low at ports known to be dominantly transhipment ports, mainly because of the frequency and intensity of smaller vessel movements between hub ports and feeder ports, which has the effect of reducing the relative importance of megaships. Ports being both gateways and transhipment hubs, such as Rotterdam, Antwerp, and Busan, thus have a lower share of mega-ship traffic. Of course, the quality of port infrastructure also plays a role, as seen with the higher percentage of mega-ship traffic in Yangshan, Shanghai's offshore hub, but also in Piraeus, where the terminal handling company COSCO obtained a concession nearly a decade ago. Last but not least, a number of smaller ports exhibit a higher share of mega-ship traffic mainly in Europe, such as Gdansk, Aarhus, Bremerhaven, Felixstowe, but also in Asia with the case of Dalian. Such effects as mainly conjectural and supported by specific windows of opportunity, such as in Gdansk, the largest port of the Baltic Sea in in the mid-2010s, which stands out due to its recent inclusion in the services of the G6 alliance (OECD-ITF, 2016

Table 1 : Top 25 ports in the world handling mega-ships, 2016

 1 ). Source: own elaboration based on Lloyd's List Intelligence data

		Ningbo	30.9 Cai Mep	23.9
	18	Piraeus	26.0 Dunkirk	23.2
	19	Algeciras	16.9 Rotterdam	22.5
	20	Dalian	27.2 Khor Fakkan	22.2
	21	Colombo	13.6 Xiamen	22.0
	22	Jebel Ali	7.7 Evyap	21.9
	23	Hong Kong	3.1 Hamburg	21.3
	24	King Abdullah Port	46.0 La Spezia	20.7

For a review about the variety of all sorts of shipping data, seeDucruet (2017)

For more information on the creation of the grid with an application to the Mediterranean basin, seeDucruet and Bunel (2017)

Acknowledgements

The research leading to these results has received funding from the European Research Council under the European Union's Seventh Framework Programme (FP/2007-2013) / ERC Grant Agreement n.

[313847] "World Seastems". For more information: www.world-seastems.cnrs.fr


Séverin L Nadji

Michel Lequime
email: michel.lequime@fresnel.fr

Thomas Begou

Cihan Koc

Catherine Grèzes-Besset

Julien Lumeau

In-situ interferometric monitoring of optical coatings

We present a new method for the in situ measurement of the amplitude and phase of the reflection coefficient of a plane substrate installed in a mechanical holder rotating at high speed (120 turns per minute) during the deposition of an optical thin-film stack. Our method is based on digital holography and uses a self-referenced scheme to cancel the effects of the severe constraints generated by the vibrational and thermal environment of the deposition machine.

Introduction

The realization of complex optical filtering functions requires perfect control of the deposition process as well as accurately monitoring the thicknesses of the deposited layers in real time. Existing techniques are based on physical or optical methods, the principle consisting in the latter case of following the change in transmittance T (or the reflectance R) of a witness sample during the stack deposition. If this measurement is made at a single wavelength, one talks of monochromatic monitoring, otherwise when this measurement is made over a wide spectral range, the method is defined as broadband monitoring [START_REF] Tikhonravov | Optical monitoring strategies for optical coating manufacturing[END_REF].

All these optical methods are based on intensity measurements, and consequently the phase information, which is a critical parameter in a multiple-wave interference phenomenon such as that involved within a multi-layer stack, is totally lost. Some studies [START_REF] Lee | Optical monitoring and real time admittance loci calculation through polarization interferometer[END_REF][START_REF] Wu | Optical Admittance Monitor through a Dynamic Interferometer[END_REF][START_REF] Wu | Multilayer thin-film inspection through measurements of reflection coefficients[END_REF] have shown that it is possible to make an interferometric measurement of the stack properties in the noisy environment of a deposition machine by using Dynamic Interferometry ® . This technique, developed and patented by 4D Technology [5], implements a micro-polarizer pixelated camera to achieve the instantaneous acquisition of four different phase states of the field reflected by the sample [START_REF] Brock | Dynamic Interferometry[END_REF]. It is clearly a very efficient solution, but quite expensive and requires a contractual agreement with 4D Technology to use their patented sensor (PolarCam™) within an interferometric set-up. Moreover, a tunable retarder is used together with a low coherence source to record only the result of the interference between the fields reflected by the coated front face and by the uncoated rear face, used here as a reference surface [START_REF] Lee | Optical monitoring and real time admittance loci calculation through polarization interferometer[END_REF]. This means that the measurement is sensitive to any phase variations between these two surfaces, such as that induced by a temperature change in the witness glass.

These are the main reasons why we have chosen to develop an alternative approach using a self-referenced interferometric configuration, in which the phase information is obtained by digital holography processing [START_REF] Picart | Digital holography[END_REF]. In section 2, we first present the main characteristics of the deposition machine, the self-referenced scheme we implemented for the substrate as well as the structure of the interferometric measurement set-up. Section 3 is devoted to the presentation of a theoretical model that predicts the temporal change in the data provided by the interferometric set-up throughout the deposition of single layers (high index and low index), but also provides a detailed description of the data processing used to extract the modulus and phase of the reflection coefficient. In Section 4, we present the main experimental results obtained with this interferometric configuration and we analyze the agreement between the recorded phase information and the predictions of our theoretical model. Finally, we conclude and define the possible applications of our method (Section 5).

Materials and Methods

Deposition machine

The deposition machine uses a dual ion beam sputtering technique (DIBS) to obtain dense layers of high purity oxides. Figure 1 illustrates the inside of the vacuum chamber showing three planar targets of silicon, tantalum, and hafnium mounted on a rotating mechanism. Each material is deposited by sputtering the surface of the corresponding target with a highly energetic argon ion beam (not visible in the picture) in a reactive oxygen atmosphere. During the growth of the layers, a second lower-energy ion beam, mixing argon and oxygen, is directed towards the sample to adjust the stoichiometry of each oxide layer (SiO 2 , Ta 2 O 5 , HfO 2 ) and improve their packing density. The sample to be coated is a plane silica window 25 mm in diameter, 1 or 2 mm thick, installed in a rotating substrate holder, as shown on the left of Figure1. The rotary movement of the substrate (rotation speed 120 turns per minute) is used to improve the uniformity of the layers deposited at the bottom surface of this sample. This sample can be illuminated with a collimated probe beam from the top of the machine through the entrance window, and this beam is shifted sideways by a periscope. This probe beam is thus fixed relative to the machine, which means that it is necessary to synchronize any transmission or reflection measurement with the rotation of the substrate holder [START_REF] Lequime | Determination of the optical constants of a dielectric layer by processing in situ spectral transmittance measurements along the time dimension[END_REF].

Measurement system

The design of the interferometric measurement system must take into account three main constraints, namely:

1. a continuous displacement of the sample with respect to the machine frame, which can be caused by thermal expansion of the chamber and wobbling of the substrate holder, 2. the vibration of the machine caused by the vacuum pump, 3. the presence of a parasitic signal due to the reflection of the probe light beam from the top face of the substrate.

To overcome the first constraint, we insert a very thin plane steel ring between the bottom face of the substrate and the substrate holder, as shown in Figure 2. This annular stop prevents the deposition of any material onto the outer zone of the substrate, and this uncoated circular crown can be used as a local reference for the interferometric measurement of the layer thickness deposited over the central zone. Consequently, the result of a measurement is the difference ∆φ between the phase values recorded at the center (φ C ) and in the outer annular zone (φ A ) respectively of the substrate. To solve the vibrations problem, we used a powerful light source working in triggered mode, synchronized to the substrate holder rotation (2 Hz) and emitting a very short pulse (10 µs), allowing an instantaneous interference state when the witness sample is centered on the probe beam to be recorded. Spurious interference signals are suppressed in the same way as that implemented by Lee et al. [START_REF] Lee | Optical monitoring and real time admittance loci calculation through polarization interferometer[END_REF], namely the use of a low coherence source.

We installed on the top of the deposition machine a high stiffness optical bench holding a Michelson type interferometer with one arm formed by the periscope-substrate assembly, as shown in Figure 3. A low coherence source (superluminescent diode, central wavelength λ 0 = 830 nm, spectral bandwidth ∆λ = 17 nm) is coupled into a single mode fiber whose output end is placed in the focal plane of an aspherical lens with a focal length of 150 mm. The collimated beam provided by this assembly is divided into two perpendicular arms by a cube beam splitter. The light beam transmitted by the splitter (reference beam) is reflected by a plane mirror mounted on a motorized translation stage, while the beam reflected downwards by the same beam splitter (probe beam) illuminates the whole surface of the witness substrate after passing through the periscope. Both reflected beams are recombined by the splitter cube and detected by a low-noise 14-bit CCD camera with 1600 × 1200 pixels of 7.4 µm square. Finally, a low-distortion photographic lens is used to image the bottom surface of the witness sample on this CCD matrix.

A small tilt is introduced between the two end-mirrors of the Michelson interferometer, in order to produce a quasi-parallel fringes pattern at the surface of the CCD, as shown in Figure 4.

To ensure a good quality recording of this fringe pattern by the CCD matrix, it is first necessary for the pixel size p to be not greater than one sixth of the fringe period P, i.e.

p P 6 = λ 0 12 θ ( 1
)
where θ is the instantaneous value of the tilt angle between the two end-mirrors. This leads a maximum tilt angle of approximately 10 milliradians (0.6 degree) to be specified between the two end-mirrors of the Michelson interferometer. However, this fringe pattern must also be recorded with good contrast, which requires that the changes of the relative axial position between these two end-mirrors be less than half the coherence length l c of the source, i.e.

δz l c 2 = λ 2 0 2∆λ ∼ 20 µm (2)
To relax this axial stability constraint up to 100 µm, while maintaining the benefits of the low coherence interferometric scheme, an additional bandpass filter (central wavelength 830 nm, spectral bandwidth 3.2 nm) is placed in front of the CCD camera; furthermore, this filter reduces the amount of parasitic light seen by the CCD matrix.

Theoretical model

Reflection coefficient

As emphasized in Section 2.2, the result of a measurement is the difference between the phase values recorded in the central zone C and the annular zone A, i.e. to a first approximation,

∆φ = φ C -φ A = ρ c -ρ A ( 3
)
where ρ is the phase of the reflection coefficient r, defined as Figure 5 gives a schematic view of the measurement method. For the reference zone A, the reflection coefficient is given by:

r = √ R e iρ (4)
r A = n s -1 n s + 1 (5)
where ñs is the effective index of the substrate. Consequently: ρ A = 0.

Similarly, for the central zone C, the reflection coefficient is given by [START_REF] Macleod | Thin-Film Optical Filters[END_REF][START_REF] Amra | Electromagnetic Optics in Thin-Film Coatings[END_REF]:

r C = ñs -Y 0 ñs + Y 0 ( 6
)
where Y 0 is the complex admittance of the bottom interface of the substrate (i.e. the top interface of the stack). For example, in the case of a single layer, we have

r C = n 1 (n s -n v ) cos δ 1 -i(n s n v -n 2 1 ) sin δ 1 n 1 (n s + n v ) cos δ 1 -i(n s n v + n 2 1 ) sin δ 1 (7)
which leads to the following expression for the phase ρ C :

ρ C = arctan 2n s n 1 (n 2 1 -n 2 v ) sin 2δ 1 (n 2 1 + n 2 v )(n 2 s -n 2 1 ) + (n 2 1 -n 2 v )(n 2 s + n 2 1 ) cos 2δ 1 (8)
Figure 6 shows the variation of the phase difference ∆φ = ρ C -ρ A versus the thickness of the deposited layer, on the left for SiO 2 and on the right, for Ta 2 O 5 . Note that the non-linearity of this relation increases with the mismatch between the refractive index of the layer and that of the substrate. Note also that the deposition of a silica layer onto a silica substrate can be monitored using this interferometric approach even if their refractive indices are identical; this would be impossible in the case of intensity monitoring.

Data processing

Quite generally, the signal recorded by the CCD matrix is described by the following relation:

S(x, y) = S 0 (x, y) + S 1 (x, y) cos 4π λ (αx + βy) + Φ(x, y) (9)
where α and β are the components of the tilt angle along x and y axes.

Figure 7a shows a false color representation of this fringe pattern when the central zone of a substrate is coated with a 90 nm thick Ta 2 O 5 layer. The surface flatness error of this substrate is roughly λ, causing a slight deformation of the fringes, almost imperceptible on the illustration.

A 2D fast Fourier transform is applied to this digitized signal, resulting in three separate orders (see Figure 7b for a false color representation of this Fourier spectrum in log scale), in accordance with the following expression:

S( f x , f y ) = S 0 ( f x , f y ) + S + ( f x -2α/λ, f y -2β/λ) + S -( f x + 2α/λ, f y + 2β/λ) ( 10
)
where S is the Fourier transform of S and where S ± (x, y) is defined by:

S ± (x, y) = 1 2 S 1 (x, y)e ±iΦ(x,y) (11)
Windowing is then applied to this spectrum around the first order (see Figure 7c) and the filtered data are re-centered on the spatial frequency origin (see Figure 7d). Note that the quality of this windowing requires that the ±1 orders are sufficiently far from the central zero order (tilt angle θ > 5 milliradians). An inverse 2D fast Fourier transform is applied to this re-centered signal to recover the function S + (x, y). A modulo 2π determination of the phase Φ(x, y) is given by while the modulus of S + (x, y) is simply equal to 2S 1 (x, y) (see Figs.7e and 7f respectively). Note in Figure 7f the deformation of the phase map levels caused by the initial surface flatness error of the substrate. Finally this phase, modulo 2π, is transformed into an absolute value, via a phase unwrapping procedure based on the algorithm developed by Herraez et al. [START_REF] Herraez | Fast two-dimensional phase unwrapping algorithm based on sorting by reliability following a noncontinuous path[END_REF]. Some examples of phase maps recorded before and after unwrapping are shown in Figure 8 By subtracting the unwrapped phase map recorded without coating from that recorded during deposition, the influence of the flatness error of the substrate is canceled (see Figure 8e). However, to simulate the effects of vibration, the tilt of the substrate is modified in our modeling program before each new acquisition: this explains the graded color changes observed in the central and annular zones. By subtracting the tilt value determined over the reference annular zone, this effect is canceled and the phase difference ρ C -ρ A (see Figure 8f) is finally obtained, as expected.

φ(x, y) = arctan [S + (x, y)] [S + (x, y)] ≡ Φ(x, y) (mod 2π) (12)

Experimental results

The left of Figure 9 shows an overall view of the deposition machine with, on the top, the interferometric measurement system, and to the right of the same Figure, a closer view of the upper part of this experimental set-up. Note that the reference arm of the Michelson interferometer is folded using two plane mirrors at 45 degrees to satisfy dimensional constraints.

Preliminary characterizations

Before applying this interferometric set-up to monitoring the deposition of a single layer, some preliminary tests were carried out to quantify its intrinsic performance.

The first test consists in using the motorized translation stage to scan the axial position of the reference mirror over a distance that allows the intensity of the echoes respectively associated with the reflection from the front face and from the rear face of a 2 mm thick bare silica substrate located in the substrate holder to be recorded. The result of this measurement is shown in Figure 10. Each maximum of visibility corresponds to a position of the reference mirror for which interference with an optical path difference of zero occurs. Consider the blue dots in the graph of Figure 10, recorded when the deposition chamber is filled with air: the left maximum (z = 7.632 mm) corresponds to the reflection from the top face of the substrate, while the right maximum (z = 10.716 mm) is associated with the reflection from its bottom face. The distance between these two maxima, i.e. 3.084 mm, is in good agreement with the optical thickness of the substrate (2 mm). When the same recording is made under vacuum (red dots graph of Figure 10), the positions of both maxima are slightly shifted toward the low z values, in accordance with the refractive index decrease (approximately 3 × 10 -4 ) caused by an air-to-vacuum transition: indeed, this change causes a slight decrease in the optical path length of the Michelson arm whose substrate is the end-mirror. However, the distance between these two echoes is not changed (10.413 -7.329 = 3.084 mm), since the optical thickness of the substrate is not affected by this air-to-vacuum transition. Finally, note that the full width at half maximum of each of these echoes is sufficiently narrow to guarantee that there is no overlap.

The second test consists in recording the time fluctuations of the phase difference ∆φ measured over approximately 5 minutes on the same bare silica substrate in both static and dynamic configurations, i.e. with and without rotation of the substrate holder. The results are summarized in Tab. estimate the accuracy in term of deposited thickness, we have to divide these data by 2 (reflective configuration) and take into account the refractive index of the layer: this leads for instance to a measurement noise σ d of about 2 nm in the case of a Ta 2 O 5 layer. The two top graphs correspond to the phase and intensity maps recorded just before deposition. The substrate holder is rotating and the deposition chamber is under vacuum. The two ion beams (sputtering and assistance) are ready to be activated. The two bottom graphs correspond to the same maps, but after deposition of a 50 nm thick Ta 2 O 5 layer. The uniformity of the phase data recorded over both the central and annular zones, but also the level variation of the intensity recorded over the surface of the sample, reveal the Gaussian shape of the probe beam as well as its relatively narrow spectral bandwidth (speckle pattern). To overcome this difficulty, the modulus of the reflection coefficient is calculated using the following formula:

Monitoring the deposition of a single layer

|r | = S 1 C S 1 A × S 1 A,0 S 1 C,0 |r | 0 = S 1 C S 1 A × S 1 A,0 S 1 C,0 • n s -1 n s + 1 ( 13
)
where the quantities with subscript 0 correspond to the values recorded just before the start of deposition.

Figure 12 shows the change in the modulus and phase of the reflection coefficient with respect to time during the deposition of a half-wave silica layer onto a silica substrate. The red dots correspond to measurement results, namely the modulus of r and its argument ρ divided by π. The green dots also correspond to experimental results, but the color change is used simply to identify the acquisitions performed before the start of the layer deposition. Moreover, by inverting relation [START_REF] Brock | Dynamic Interferometry[END_REF], the admittance Y 0 can be derived from the reflection coefficient r, i.e.

Y 0 = n s 1 -r 1 + r (14)
The result of this calculation is presented on the right of Figure 12 as a trajectory in the complex plane (admittance locus). Finally, on these three graphs the blue line corresponds to a theoretical fit, based on relation [START_REF] Picart | Digital holography[END_REF], in which the only free parameters are the refractive index of the layer (n 1 ), the deposition rate (v), assumed constant, and the time value t 0 corresponding to the start of the layer deposition. Consequently, we have:

d 1 (t) = 0 if t t 0 v(t -t 0 ) if t t 0 (15)
The fitting process provides the following results: t 0 = 80 s, v = 0.374 nm/s, and n 1 = 1.4865, the agreement between experimental data and theoretical fit is excellent. Moreover, the deposition rate and the refractive index are comparable with those obtained a few months ago [v = 0.3364 nm/s, n 1 = 1.4837] on the same deposition machine using another method [START_REF] Nadji | Use of a broadband monitoring system for the determination of the optical constants of a dielectric bilayer[END_REF].

The results obtained with the same approach during the deposition of a half-wave tantala layer on a silica substrate are shown in Figure 13. Again, the agreement between experimental results Time Fig. 13. Experimental data recorded during the deposition of a half-wave tantala layer on a silica substrate (see text for more details). and theoretical fit for the modulus of the reflection coefficient is very satisfactory, but there is also a slight discrepancy in the phase term (about 0.25 radians). This small difference is notably visible on the admittance locus graph (right of Figure 13) where the center of the blue circle clearly does not appear to be in the right place, even though its radius seems correct. Adding 0.25 radians to the theoretical phase values shifts the model data (thin black circle) to the right place with respect to the experimental data. We still have no satisfactory explanation for this discrepancy. Note that the refractive index and deposition rate values (respectively, 2.1137 and 0.255 nm/s) are in good agreement with the results of previous measurements [START_REF] Lequime | Determination of the optical constants of a dielectric layer by processing in situ spectral transmittance measurements along the time dimension[END_REF].

Discussion and conclusion

Discussion

This interferometric approach allows the growth of a single layer to be monitored through the temporal change of the admittance Y 0 in the complex plane. The trajectory shape shows that the accuracy of the deposition cut should be nearly independent of the layer thickness.

To confirm this preliminary conclusion, we consider multilayer stacks, such as a quarter-wave mirror, as illustrated by Figure 14. High index layers (tantala) correspond to red circles, low index layers (silica) to blue circles; green (respectively yellow) dots are used to identify the start (respectively the end) of the deposition. Note that phase variations recorded during the deposition of a layer become lower and lower, which can progressively decrease the signal-to-noise ratio of the measurements for quarter-wave mirrors with large number of HL pairs. If we now consider the case of an 8-layer antireflective coating (see Figure 15) that includes some very thin layers with thicknesses less than 25 nm, we see that the phase variations are mostly linear throughout the stack deposition, while the dimensions of the admittance locus are approximately the same as those recorded during the deposition of a half-wave silica layer. The same conclusion can be formulated for monitoring the adaptation layer inserted between the two cavities of a Fabry-Perot bandpass filter: the use of interferometric monitoring provides a real advantage with respect to methods based only on intensity measurements.

Conclusion

We have demonstrated that the use of an interferometric scheme can provide useful information for the in situ monitoring of the deposition of transparent layers. The implementation of a self-referenced scheme allows accurate measurements to be obtained despite the severe constraints created by the thermal and vibrational environment, and nothing appears to prevent this method from being extended to the monitoring of thin absorbing or metallic layers. However, it would be necessary to add a motorized sub-assembly using e.g. a Risley prism pair to provide a dynamic alignment capability of the probe beam with respect to the substrate (as stressed above, the tilt angle between the reference mirror and the substrate must remain between 0.005 radians and 0.01 radians). Moreover, it is surely possible to implement simultaneously optical broadband monitoring (we have successfully carried out some preliminary tests in this direction) so as to mix intensity and phase measurements in this way.

In addition, the use of low coherence interferometry allows easy implementation of some other on-line measurements, such as in situ stress monitoring (measurement of the radius of curvature of the rear face of the substrate), or estimating the uniformity of the deposited layers and even a real time measurement of the temperature of the substrate.

Fig. 1 .

 1 Fig. 1. The DIBS deposition machine (on the left, schematic view of the substrate configuration; on the right, picture of the inside of the machine).

Fig. 2 .

 2 Fig. 2. Principle of the substrate self-referencement.

Fig. 3 .Fig. 4 .

 34 Fig. 3. Schematic representation of the interferometric set-up installed on the top of the DIBS vacuum chamber.

Fig. 5 .

 5 Fig. 5. Schematic view of the measurement method.

Fig. 6 .

 6 Fig. 6. Theoretical variation of the phase difference ∆φ versus the thickness of the deposited layer (on the left, silica; on the right, tantala).

Fig. 7 .

 7 Fig. 7. Numerical modelling of the different steps used for the data processing (see text for more details).

Fig. 8 .

 8 Fig. 8. False color representation of the phase maps used to extract the phase difference between the central zone and the reference annular zone (see text for more details).

Fig. 9 .

 9 Fig. 9. Photographs of the experimental set-up (on the left, overall view of the deposition machine; on the right, a closer view of the upper part of the interferometric set-up).

Fig. 10 .

 10 Fig. 10. Recording of the axial position of the echoes associated with the reflection from both faces of the substrate (blue dots, in air; red dots, in vacuum).

Figure 11shows two examplesFig. 11 .

 11 Figure 11shows two examples of results obtained during the deposition of a Ta 2 O 5 layer.

Time

Fig. 12 .

 12 Fig. 12. Experimental data recorded during the deposition of a half-wave silica layer onto a silica substrate (see text for more details).

Fig. 14 .

 14 Fig. 14. Theoretical data recorded during the deposition of a quarter-wave mirror (tantala/silica) onto a silica substrate (see text for more details).

Fig. 15 .

 15 Fig. 15. Theoretical data recorded during the deposition of an 8-layer antireflective coating onto an N-BK7 substrate.

Table 1 .

 1 1, where ∆ is the optical path difference associated with ∆φ [∆ = ∆φ × (λ/2π)]. To Intrinsic Performance of the set-up in static and dynamic configurations

	Configuration	∆φ (radians) σ ∆φ (radians)	∆ (nm) σ ∆ (nm)
	static	0.005	0.015	0.7	2.0
	dynamic	0.014	0.065	1.8	8.6

Acknowledgments

This work was carried out within the LabTOP (Laboratoire commun de Traitement OPtique des surfaces), a research cooperation agreement between the Institut Fresnel (Aix-Marseille Univ, CNRS, Centrale Marseille) and CILAS Ariane Group.

Disclosures

The authors declare no conflicts of interest.


A Bourgade

F Lemarquis

A Moreau

T Begou

J Lumeau

CHALCOGENIDE-BASED ULTRA-UNIFORM NARROW BANDPASS FILTERS

Keywords:

Very large progress has been made within the fabrication method and the complexity of optical thin film filters. Coatings can now be accurate at nanometric scales even for stacks up to hundreds of layers. However, more and more uniform and performant filters are now needed. After filter fabrication the spectral performances of a filter are fixed. To overcome this limitation, materials which refractive index or thickness can be locally changed after exposure with an actinic radiation can be used.

In this paper, we present the fabrication of bandpass filters based on photosensitive optical thin films (As 2 S 3 ). Filters centered at 800 nm, with bandwidth of 1% or below and rejecting the whole silicon detector sensitivity spectral range are demonstrated. These filters are composed with single or multiple Fab-ry-Perot cavities and have a nominal uniformity of 1% over 100 mm diameter aperture. By using the photosensitive properties of the chalcogenide layers, we show that filters with uniformity below ±0.05% can be achieved. The fabricated filters are shown to have identical performances when used locally or full aperture.

INTRODUCTION

For the past couple of decades, there has been very large progress within the fabrication method and the complexity of optical thin film filters. Coatings can now be accurate at nanometric scales even for stacks up to hundreds of layers. However, more and more uniform and performant filters are now needed, requiring better and better control of the processes during the deposition. Spectral performances of a filter are related to the optical thickness of each layer and performances are fixed at filter achievement. Moreover, due to variations of the deposited thickness over the substrate aperture, the performances of the filters vary accordingly. This problem is particularly important in case of narrow bandpass filters. Actually, it can be shown [START_REF] Lequime | Toward tunable thin-film filters for wavelength division multiplexing applications[END_REF] that the relative variation of the central wavelength (λ) of a bandpass filter is directly related to the relative variation of the thickness (nt) of its cavity:

(1)
Where κ is a factor taking into account the phase dispersion derivative of the mirrors that we considered as equal to one in this paper. As of today, the best deposition techniques allow securing a close to 0.1% change of the thickness over a 100 mm aperture [START_REF] Begou | Complex optical interference filters with stress compensation for space applications[END_REF]. This non uniformity results in variation of the central wavelength of 1 nm for a filter centered at 1 µm. But most of the standard deposition techniques based on evaporation by sputtering are generally resulting in uniformities within the 1%-range such as it is not possible to fabricate large aperture filters with bandwidth smaller than 10 nm.

In this paper, we propose to overcome this limitation by using materials which optical thickness can be changed after deposition. The concept of such an approach was introduced some time ago [START_REF] Lequime | Laser trimming of thin-film filters[END_REF] and basic proof-of-concept was published about 10 years ago [START_REF] Shen | Photosensitive post tuning of chalcogenide Te20As30Se50 narrow bandpass filters[END_REF]. This approach relies on the fabrication of bandpass filters made of photosensitive materials in order to locally correct the central wavelength of the filter using photo-induced refractive index change. However, in ref. [START_REF] Shen | Photosensitive post tuning of chalcogenide Te20As30Se50 narrow bandpass filters[END_REF], only small 5×5 mm² uniform bandpass filter was demonstrated. In this paper, we present a thorough analysis of the fabrication of large aperture narrowband pass filters at 800 nm. We first present a characterization of the photosensitivity of thin As 2 S 3 films and then the use of this material for the fabrication of high performance bandpass Fabry-Perot filter. We then present a method for automatic local correction of a bandpass filter and demonstrate a highly uniform 60×60 mm²-square filter with 5 nm bandpass and ±0.1% fluctuations of the central wavelength [START_REF] Bourgade | Large aperture, highly uniform narrow bandpass Fabry-Perot filter using photosensitive As 2 S 3 thin films[END_REF].

SINGLE CAVITY BANDPASS FILTERS

For the development and manufacturing of photosensitive optical thin films, we used chalcogenide glasses such as As 2 S 3 also known as AMTIR-6. This glass is known for its broad transparency in the near and far infrared (AMTIR stands for Amorphous Material Transmitting Infrared Radiations) and has the unique characteristic of being photosensitive, i.e. some of its opto-geometrical properties can be modified after exposure to actinic radiation. This characteristic has already been demonstrated in linear regime [START_REF] Hisakuni | Laser-induced persistent self-focusing in As 2 S 3 glass[END_REF][START_REF] Tanaka | Photoinduced Bragg gratings in As 2 S 3 optical fibers[END_REF] and non-linear one (two photons absorption) [START_REF] Meneghini | As 2 S 3 photosensitivity by two-photon absorption: holographic gratings and self-written channel waveguides[END_REF]. In previous research, it has been shown that the photo-induced refractive index change can go up to 0.016 [START_REF] Hu | Resonant cavity-enhanced photosensitivity in As 2 S 3 chalcogenide glass at 1550 nm telecommunication wavelength[END_REF]. Many applications have also been demonstrated with such a material including waveguides for example [START_REF] Hisakuni | Laser-induced persistent self-focusing in As 2 S 3 glass[END_REF][START_REF] Vallée | Real-time observation of Bragg grating formation in As 2 S 3 chalcogenide ridge waveguides[END_REF]. This material and its photosensitivity has also been used to create gratings and lenslets on the surface of those films with above band gap illumination [START_REF] Ramachandran | Fabrication of holographic gratings in As 2 S 3 glass by photoexpansion and photodarkening[END_REF][START_REF] Hisakuni | Optical fabrication of microlenses in chalcogenide glasses[END_REF]. But, to the best of our knowledge, no optical quality coatings compatible with the manufacturing of complex optical elements have ever been fabricated. The chalcogenide layers and thin film stacks were produced using electron beam deposition in a Bühler SYRUSpro710 machine. The whole deposition was monitored by optical monitoring using a Bühler OMS 5000 system and deposition rate was controlled with quartz-crystal monitoring system. The material used to deposit is a bulk amorphous As 2 S 3 glass blank. The residual pressure inside the vacuum chamber was around ~10 -6 mbar, and the deposition rate was set to 3Å ∕s. The chalcogenide layers were fabricated using electron beam deposition while the Bragg mirrors of the Fabry Perot Filter (made with alternated Nb 2 O 5 and SiO 2 layers) were fabricated using plasma assisted electron beam deposition. Due to the photosensitive nature of such a material, we protected the samples from any ambient light exposure by keeping them in dark boxes, except during the exposure process.

To characterize the photosensitive properties of the As 2 S 3 layers, we deposited 990 nm thick (3λ/2 at 1550 nm) single layers. Then, using a Perkin Elmer Lambda 1050 spectrophotometer, we measured the spectral dependence of the transmission before and after exposing the layer to a 20 mW LED at 470 nm (Fig. 1). One can see that after exposing the layer with a dosage of 110 J/cm², the transmission spectrum is shifted to longer wavelengths by 3.5% and the amplitude of the intensity modulation is also increased by 6.4%. Reverse engineering on these two curves [START_REF] Gao | Refractive index determination of SiO 2 layer in the UV/Vis/NIR range: spectrophotometric reverse engineering on single and bi-layer designs[END_REF] allows to show that at 800 nm, the refractive index is changed from 2.412 to 2.497 without any change of the layer thickness.

To better characterize the refractive index change kinetics, we then exposed the As 2 S 3 layer with different dosages, measured the spectral dependence of the transmission and reflection after each dosage increment and finally implemented a reverse engineering technique to extract the opto-geometrical parameters of the layer. Fig. 2 shows the evolution of the refractive index change in As 2 S 3 layer for exposure dosage from 0 to 110 J/cm². A refractive index increment as large as 0.08 at 800 nm can be induced with a dosage of ~20 J/cm². For larger dosages, a saturation of the refractive index is observed. In addition, it is worth noting that no change of the layer thickness could be detected with our reverse engineering method and this was confirmed by optical profilometer measurements. Finally, the observed refractive index changes are non-reversible and stable (at least for more than 12 months -time since we started this work) at room temperature.

We then analyzed how the As 2 S 3 layer can be integrated within a multilayer structure. We designed a narrow bandpass filter centered at 800 nm with a single cavity Fabry-Perot structure, i.e. composed of one As 2 S 3 cavity separated by two dielectric quarter wave layer-based mirrors. The materials used for the mirrors are silica (SiO 2 ) as low index material and niobia (Nb 2 O 5 ) as high refractive index material. The design of the filter is:

(HL) 4 /4 ChG / (LH) 4 (2)

where H stands for a quaterwave Nb 2 O 5 layer, L for a quarterwave SiO 2 layer and ChG for a quarterwave As 2 S 3 layer. Such a filter generates a narrow bandpass with a Full Width at Half Maximum (FWHM) of 4 nm. A prototype of this filter was fabricated within our technological platform on a 4-inch diameter glass substrate. After deposition, the filter was characterized with a Perkin Elmer Lambda 1050 spectrophotometer between 450 and 1100 nm. In Fig. 3, we overlapped the measured (blue curve) and theoretical (red curve) spectral dependence of the transmission of the filter. One can see that the overall experimental transmission curve is very similar to the theoretically calculated curve. However, one can see that the central wavelength is blue shifted while the side of the mirrors are red-shifted. This observation shows that the cavity was not perfectly matched during the fabrication. In addition, as the optical monitoring was done on a separate test glass, this error might also be due to inherent non-uniformity of the deposition. Indeed, it is well known that the thickness of the deposited layers will vary over the filter aperture resulting in a variation of the spectral performance. Such effect becomes noticeable and non-negligible with 4-inch diameter filters. In order to characterize this uniformity, we developed a dedicated optical setup (Fig. 4).

It is composed with a collimated white-light source. The 1 mm square beam is sent to the filter to be characterized and the transmitted beam is then collected within a second fiber, sent to a Wasatch Photonics VIS-NIR spectrometer and analyzed within the [400-1100] nm spectral range with a 0.6 nm spectral resolution. The sample is placed on two translation stages in order to scan the local transmission of the filter.

The local spectral dependence of the transmission was measured over 1800 1 mm² regions on a 65×60 mm² aperture. We then plotted in Fig. 5 an overlay of the measured transmission curves within the bandpass region. One can see that the filter is centered around 800 nm but the central wavelength shows some variations up to 9.2 nm, i.e. 1.1% variation of the central wavelength over the filter aperture. This dispersion illustrates the non-uniformity of the SYRUSpro machine and is within the expected performances with such a deposition system. Such a dispersion of the central wavelength makes this filter not usable over its whole aperture as the summing of the spectral perfor- SVC BULLETIN I SUMMER 2020 5 mance over its aperture will result in deteriorated spectral performances. In this work, we implemented a similar approach to that was presented by Shen et al. [START_REF] Gao | Refractive index determination of SiO 2 layer in the UV/Vis/NIR range: spectrophotometric reverse engineering on single and bi-layer designs[END_REF]. However, in order to demonstrate that this technique is reliable and predictable, we developed a pump/probe system that allowed automatic correction of the filter for each of the 1800 measured points (Fig. 4). Indeed, we added to the spectrophotometric system (probe system) an exposure system (pump system) composed with a blue LED of 20 mW emitting at 470 nm. This exposure system produces a 1 mm square spot that perfectly overlaps the transmission measurement spot.

Then, at first, we characterized the kinetics of central wavelength change versus exposure dosage on a single point. Dosage was increased up to 110 J/cm² with a 5 J/cm² step and the transmission spectrum was measured after each of these steps. By increasing the dosage, the central wavelength of the filter is red-shifted but no change of the transmission could be detected (within the precision of the measurement (±1%), confirming that the refractive index change does not induce losses within the layer. We plotted in Fig. 6 the evolution of the central wavelength as a function of the dosage. We can clearly see that the evolution of the central wavelength is similar to that of refractive index change that was measured in As 2 S 3 single layers. The kinetics are however different (4 times decrease) as the electric field distribution within the cavity at 470 nm induces a decrease of the local effective exposure dosage. A maximum change of the central wavelength of +10 nm, i.e. 1.2% can be induced with this technique. This value is therefore large enough to be able to locally correct each of the measured spectrum and make the filter uniform.

To achieve very uniform filter, we decided to center the whole filter at 801 nm with a precision of 0.1%, i.e. 10 times better than the original one. In this case, only red-shift of the filter is necessary. This case is not a limited case since if a more specific wavelength would be needed, it could be possible to adjust the fabrication procedure to guaranty that the part of the filter that has the largest central wavelength is below that targeted one.

We developed a LabVIEW program that allows to sequentially measure the local transmission, expose the sample at 470 nm and then measure the transmission again and repeating this procedure until the value of 801 ±0.8 nm (i.e. compatible with the maximum measured central wavelength on the filter) is achieved. This procedure is then repeated for each of the 1800 points.

After this point-by-point correction of the local transmission of the filter, we plotted in Fig. 7 the evolution of the central wavelength of the filter over its aperture as well as an overlay of the measured transmission spectra after correction. The variations of the central wavelength of the filter over its aperture are now 10 times lower (going from 800.8 nm to 801.7 nm) than before correction. Using this technique, the central is controlled with a ±0.05% precision range over a 65 × 60 mm² aperture. This value is equal, if not better than what can be achieved with highly uniform deposition machines [START_REF] Begou | Complex optical interference filters with stress compensation for space applications[END_REF].

In order to confirm that the filter can now be used in full aperture, we modified the set-up in Fig. 4 and expanded the illuminating beam to a diameter of 50 mm. The collecting collimator was also adapted in order to be able to collect, in the spectrometer, the whole transmitted beam and therefore be able to characterize the average transmission spectrum. This measurement was carried out before and after correction of the filter and was compared to the theoretical transmission of the filter (Fig. 8). Theoretically, the filter is expected to have a FWHM of 4 nm and a maximum transmission of 92%. The as-deposited filter presents a FWHM of ~10 nm and a maximum of transmission of ~50%. These deteriorated performances are due to the fact that with a large beam, the measured transmittance is the average of the transmission spectra over the filter aperture. Due to the non-uniformity which is 2.5× the FWHM, both the FHWM and the transmittance are highly affected. After correction, the variation of the central wavelength has been decreased by 10 times and is now only 0.25× the FWHM. The transmission of the filter becomes much closer to the theoretical one, i.e. we have the FWHM = 5 nm and the maximum transmittance = 85%).

TWO-CAVITY BANDPASS FILTERS

Most of the applications that require the use of bandpass filters generally require filters with higher performances, i.e. with a spectral profile in the bandpass which is more square and with higher rejection close to the bandpass. This can be achieved by combining several Fabry-Perot cavities in a coherent way. In order to secure that the two cavities will shift simultaneously when exposed to laser radiation, we modified the design and opted for non-quarter wave layer structures. A detailed of the design approach can be found in ref. [START_REF] Bourgade | Adjustable three-cavity narrow bandpass filters" To be submitted to Optics Letters[END_REF]. The structure of the filter is:

M6/1.85C/M6/L/M6/1.85C/M6/AR (3)
Where M6 are non-quarter mirrors and C are the chalcogenide cavity layers. Such a filter was fabricated with the Bühler SYRUSpro 710 deposition machine and the spectral performances of the filters were the characterized using a Perkin Elmer Lambda 1050 spectrophotometer. Fig. 9 shows the broadband spectral performances of the filter. Good agreement between experimental and theoretical curves can be observed.

This filter was then exposed with a green laser at 532 nm and the spectral performances around the bandpass region were measured (Fig. 10). One can see that the central bandpass can be shifted towards longer wavelength by ~1.2% without significant This correction relies on the use of photosensitive As 2 S 3 layers that are used as cavities in Fabry Perot structures. Using a relative refractive index change equal to the relative thickness change (due to non-uniformity issues) and with opposite sign, it is possible to generate highly uniform large aperture bandpass filters. We have shown a method that automatically corrects for the non-uniformity of a filter. Example of a 5 nm FWHM filter centered at 801 nm with deviation of the central wavelength less than 0.1% over 65×60 mm² squared aperture has been demonstrated. Application of the proposed approach on two-cavity Fabry-Perot filters has also been demonstrated. These results pave a way to the fabrication of large aperture, highly uniform, filters with more complex structures.

Fig. 1 -

 1 Fig. 1 -Evolution of the spectral dependence of the transmission of a 990 nm thick As 2 S 3 layer (green: as deposited layer and red: layer after exposure to a 20 mW LED at 470 nm).

Fig. 2 -

 2 Fig. 2 -Evolution of the refractive index change as a function of the exposure dosage at 470 nm.

Fig. 3 -

 3 Fig. 3 -Spectral dependence of the transmission of a (HL) 4 / 4 ChG / (LH) 4 Fabry Perot filter. Theoretical filter: red curve, experimental filter: blue curve.

Fig. 4 -

 4 Fig. 4 -Scheme of the pump/probe experimental setup used to both characterize the local spectral performances in transmission of the filter and correct the local central wavelength of the filter.

Fig. 5 -

 5 Fig. 5 -Evolution of the spectral dependence of the transmission measured on a Fabry-Perot filter. Each curve was measured on a 1 mm² spot size over a 65 × 60 mm² aperture.

Fig. 6 -

 6 Fig. 6 -Dependence on dosage of the central wavelength peak.

Fig. 7 -

 7 Fig. 7 -Spectral dependence of the transmission measured on each 1mm² spot over a 65x60 mm² aperture after correction of the filter (top) and mapping of the central wavelength of the filter after correction (bottom).

Fig. 8 -

 8 Fig. 8 -Behavior of the filter using a large beam (50mm diameter beam)

ACKNOWLEDGEMENTS

This work was partially funded by Ministry for Armed Forces (DGA) and Aix-Marseille Université.