Estelle F Rose-Koga

Kenneth T Koga

Jean-Luc Devidal

Nobumichi Shimizu

Marion Le Voyer

Célia Dalou

Max Döbeli

Celia Dalou

In-situ measurements of magmatic

Keywords: F, Cl, SIMS, EPMA, ERDA, melt inclusion

come

Introduction

The behavior of trace volatile elements (magmatic volatile components other than H2O and CO2) in magmas has inspired many scientific contributions in the past decades (e.g. Baker et al., 2005;Wallace, 2005;Behrens and Gaillard, 2006;Fischer, 2008;Aiuppa et al., 2009 and reference therein). For an extensive review of the interest of halogens in Earth Sciences see Hanley and Koga, 2018 (in special volume by Harlov and Aranovich, 2018 and reference therein). In short, the interests go from partition coefficients of F, Cl and S between minerals and melt or fluid (e.g. Dalou et al., 2012;Zajacz et al., 2012;Bernini et al., 2012;Wu and Koga, 2013;Van den Bleeken & Koga, 2015;Kusebauch et al., 2015;Joachim et al., 2017;Iveson et al., 2018), to tackling mantle source compositions of these elements using melt inclusions (e.g. Straub et al., 2003;Bouvier et al., 2010;Le Voyer et al., 2010;Helo et al., 2012;Rose-Koga et al., 2012, 2014, 2017;Métrich et al., 2014;Cabral et al., 2014;Hartley et al., 2014;Jackson et al., 2015a) or using glasses (e.g. Kendrick et al., 2012;2014a,b;2015;Jackson et al., 2015b) or minerals (olivine: Broadley et al., 2019), to measuring the diffusion rate of volatile elements to assess magma ascent rates (e.g. Llyod et al., 2014;Ferguson et al., 2016;Ruth et al., 2018) and assessing magma degassing (e.g. Bureau et al., 2000;Edmonds et al., 2001;Balcone-Boissard et al., 2010).

Bulk method for determining F and Cl contents in geological samples have improved through the years and pyrohydrolysis followed by ion chromatography have improved to reach detection limits of 0.2µg.g-1 (Michel and Villemant, 2003) even 0.1µg.g-1 (Blacone-Boissard et al., 2009), which remains a factor of 10 to 30 better than that reported later with the same analytical tehnic by Marks et al. (2016;10-20µg.g-1 for F and 20-30µg.g-1 for Cl). Here we want to compare in-situ analytical technics for F and Cl measurements because the study of smaller and smaller geological samples are requiring intercalibration and comparison of the technics to make an educated decision on which one to use to achieve the goal we have.

With recent advances of micro analytical techniques and melt inclusion studies, there is a growing body of concentration measurements of relatively volatile, light-atomicmass elements (H, B, C, F, S, Cl) in MORB glasses and primitive melts of subduction zone magmas (e.g. Sisson and Layne 1993;Métrich et al. 1999;Hauri et al., 2002;Wade et al. 2006;Le Voyer et al. 2008, 2010;Bouvier et al., 2008;Rose-Koga et al. 2012; see also references in Wallace 2005). Other volatile element, moderatly heavier, such as Br have also been successfully measured by secondary ion mass spectrometry (Cadoux et al., 2017). While C and H and to a lesser extend S are likely to suffer degassing (Dixon et al. 1995), halogens such as F and Cl (and probably Br) are often under-saturated in these primitive basaltic melts, indicating that they did not experience any degassing or fractionation event (e.g. Carroll and Webster 1994;Bucholz et al., 2013).

Among the difficulties contributing to the limited comparison of the preexisting data, was, suprisingly, the lack of published comparison of S, Cl and F measurements of standards between the two most used in situ analytical procedures: electron microprobe (EPMA) and the ion probe (SIMS). Recently, a F, S and Cl comparison between literature data obtain by EPMA and by SIMS (Le Voyer et al., 2019) concluded that inter-laboratory comparisons agreed within 10% for F and to a variable degree for S and Cl, and propose a quality controlled published-data summary table ( Electron microprobes perform microanalysis of volatile elements, but the high detection limits of this technique (tens to hundreds of ppm) place limitations on many volatile studies (cf. Devine et al. 1995 and references therein). After a study improving the F measurement by EPMA (Zhang et al., 2016), the inter-calibration of EPMA and SIMS is timely and echoes conference abstracts about this recurrent subject (e.g. Rose-Koga et al., 2008;Guggino and Hervig, 2010). This will be the basis to push further the investigations for example of the degassing processes via experimental approach and in situ melt inclusion studies. Also it is noted that F, S, and Cl are among elements that LA-ICPMS can not measure (for measurements of major elements in melt inclusions by LA-ICPMS and intercalibration with EPMA and SIMS, see Pettke et al. 2004) and therefore their in-situ measurements in small objects such as melt inclusions rely solely on the development of their measurements by EPMA and SIMS, and their intercalibration.

Volatile-rich magmas are generally those of subduction zones. The current consensus is that arc magmatism is triggered by a flux, of either a volatile-rich fluid, or a silicate melt, derived from the subducting lithosphere (e.g. Gill 1981;Tatsumi and Eggins 1995), or a fluid somewhere between the two and termed supercritical fluid (Shen and Keppler 1997;Bureau and Keppler 1999;Stalder et al. 2000). A large number of observations on isotopic compositions of arc lavas attest the necessity of slab flux in the constitution of lavas (e.g. Tatsumi and Eggins, 1995;Hanyu et al., 2012;Narvaez et al., 2018). Therefore, the nature of this flux and its interaction with solid mantle or with the subducting slab determines the element fractionation processes characteristic of this geodynamic setting (e.g. for Iwate volcano, Japan; Rose-Koga et al., 2014).

In this paper we measure F, S and, Cl by SIMS on a set of 6 glasses from the WHOI standard-set (ALV519-4-1, ALV1654-3, ALV1649-3, GL03-D51-3, GL07-D52-5, EN113-46D-2) to create 3 working curves. We use our 3 SIMS working curves to compare our SIMS measured values of 8 MPI DING glasses (ML3B-G, KL2-G,

StHs6/80-G, GOR128-G, GOR132-G, ATHO-G, T1-G, KE12
) and of 2 basaltic standards (VG2 and VG-A99) with our EPMA values. We also report independent absolute F and Cl values from elastic recoil detection analysis (ERDA) of three MPI DING glasses (KE12, ATHO-G and KL2G) which independently anchors our calibration curves. Finally, we give an example of application for EPMA and SIMS measurements of Cl and F in melt inclusions.

Standards and glass samples

All glasses and standards used have already been well documented elsewhere, and we summarize here the essential points. The set of 6 basalt samples used in this study for the SIMS analysis come from several sources ( et al. 1991;Thordarsson et al. 1996;Thornber et al. 2002).

Sulfur and chlorine

Because sulfur speciation (S 6+ or S 2-) induces changes in the SK spectral position (Carroll and Rutherford 1988), prior to sulfur concentration measurement and for every sample, the SK peak maximum was first located by using the regular automatic routine of the Cameca SX 100 software. Then, if the measured peak position differs from the one of the standard, the new value is changed in the analysis setup.

The selection of the diffraction crystals is driven by the achievements of the highest peak counts to reach very low detection limits and by looking at the region of the spectrum with no interfering peaks. Thus, chlorine and sulfur were analyzed successively by using a Large pentaerythritol (LPET) crystal.

Fluorine

The case of fluorine is more complex. This element can be measured either with a W/Si multilayer crystal (PC1) or with a thallium acid phthalate crystal (TAP).

Multilayer crystal allows high precision and accuracy measurements together with low detection limits. Unfortunately, for iron-bearing minerals or glasses, the FK peak is strongly overlapped by the shoulder of a strong FeL line. Different studies (Todd 1996 ;Witter and Kuehner 2004) have proposed an electron microprobe method for analyzing F in Fe-bearing minerals and glasses using multilayer crystals that overcomes the spectral interference. This method is based on the linear relationship existing between the iron concentration of fluorine-free minerals (olivine and pyroxenes essentially) and the number of counts at the FKpeak position in the same fluorine-free minerals. Thus, the FeL contribution (i.e. the background) can be easily deduced and quantified from the total iron concentration of the sample and subtracted from the bulk FKa peak counts. However, the calibration curve of this model is only found for the analysis of Fe 2+ -bearing minerals. In transition metals of the first row, the L-spectra exhibit peak position shifts as a function of the oxidation state (Fialin et al. 2001(Fialin et al. , 2004)). The omission of the self-absorption induced shift of the L peak between Fe 2+ and Fe 3+ could lead to the overestimation of the background counts at FKa peak position and thus to an underestimation of the fluorine content.

The correction method established by Witter and Kuehner (2004) should be only applied for pure Fe 2+ -bearing minerals and glasses. In order to overcome this problem, we analyzed fluorine using TAP diffraction crystals although the detector is significantly less efficient than PC1. To improve its counting statistics (precision and detection limit), fluorine was measured simultaneously on 3 spectrometers according to the Cameca multi-spectrometers counting routine. On top of the choice of the detector, we tested CaF 2 and Durango apatite standards for F calibration, and concluded that CaF 2 provides generally consistent results, most likely due to known F X-ray excitation issue of apatatite (Stromer et al., 1993).

The challenge with traces elements analysis in glass is to find a compromise between low detection limit, i.e. the used of high beam current, long counting time, and limited beam damages. Volatile loss during the analysis is minimized through the used of a protocol derived from the CSIRO-trace routine (Robinson and Graham 1992). The total counting time (peak and background) for a single analysis is 40 sec and is divided as follow: 10 sec on peak and background for chlorine and sulfur but 60 sec on peak and background for fluorine (20 sec per spectrometer). Low detection limit is achieved by increasing the number of analysis on the same point, thus by improving the singal-to-noise ratio. After each analysis, the beam is shielded for 20 sec allowing the sample to cool down. Total volatiles concentration is calculated from the sum of the counts from the successive iterations. With 15 kV accelerating voltage and 80 nA beam current, for a total Cl and S peak counting time of 100 sec and 600 sec for F. Typical detection limits for F, Cl, and S were 150, 50 and 50 µg.g -1 , respectively.

SIMS analysis

Sample preparation for SIMS

The standards are mounted in high purified indium metal (e.g. Hauri et al. 2002;Le Voyer et al. 2008) in a 1 inch diameter aluminum ring, put in ultrasound in pure ethanol then in distilled water for 10 minutes, respectively. Indium is used because epoxy can contain significant amounts of volatiles that can degass during the analysis and increase the background signal. The mount is dried carefully in an oven overnight. The mount is finally gold coated before analysis and kept overnight in high vacuum (low 10 -8 torrs) until being inserted in the sample chamber.

Method

The measurements for the working curve calibrations were done on a set of 6 glass standards (Table 1), on the ion probe Cameca 1280 of Woods Hole Oceanographic Institution (MA, USA). We used a Kohler illumination with a primary beam current of 1.5 nA Cs + primary positive beam, and negatively charged secondary ions were extracted through a nominal accelerating potential of 10 kV. Due to implantation of Cs + ions and extraction of both negatively charged secondary ions and electrons, positive charging of the sample surface must be compensated with the use of an electron flood gun which delivers electrons to the sample surface. The isobaric interference were filtered by an energy slit opening at 50 eV and the contrast aperture at the cross over was large (400 µm). The entrance and exit slits are closed to achieve a mass resolution of M/M=5800.

We presputtered the samples surface during 180 seconds while applying a raster of 30×30 µm. The field aperture (of 8000) corresponds to an area of 15×15 µm, is inserted into the image plane. This means that only the ions originating from the central 15 µm of the flat-bottomed sputtered-crater are admitted into the mass spectrometer. The elimination of stray ions sputtered from the crater walls and desorbed from the sample surface results in very low volatile backgrounds (routinely about 0.05-0.1 counts per second for the counting system at half mass positions with the primary beam and the electron gun on). We counted 8 sec on 19 F, 5 sec on 30 Si, 5 sec on 32 S and 8 sec on 35 Cl. One analysis was composed of 2 blocks of 10 cycles and took less than 15 min per spot. Intensities of 19 F, 32 S and 35 Cl were collected cyclically by an electron multiplier, processed through pulse-counting electronics and normalized to 30 Si for concentration calculations.

Calibration

Earlier studies that have involved Cs + beam were performed on small format SIMS (Cameca 6f, Hauri et al. 2002). But hydride interferences, such as SH -, are difficult to effectively eliminate using the energy filtering technique (Shimizu et al.

1978) available on small format Cameca instruments (e.g., IMS 3f/4f/5f/6f). The high mass resolution of the SIMS 1280 of WHOI is required to eliminate the 34 S 1 H interference on 35 Cl (MRP>5120) without giving up transmission significantly (Fig. 1c). The SIMS calibration curves for F, S and Cl are shown in Fig. 2. They are regressions of ion probe signals (x-axis) compared to known EPMA concentrations (y-axis). The former is the intensity ratio of two elements times the SiO2 concentrations of each standards, the numerator of the ratio being the element of interest and the denominator is a matrix element common to all samples (e.g.

19 F/ 30 Si). Typically, here 19 F, 32 S and 37 Cl are normalized against 30 Si. This provides a robust analysis little influenced by primary beam fluctuations or by ionization efficiency changes owing to matrix effects (Shimizu and Hart, 1982). In fact, the calibrations for F, S and Cl are free of significant matrix effects. The calibration curve is determined at the beginning and at the end of each session to assure no significant drift has taken place.

Detection limit

With the calibration curves of the standards, one usually attributes the Yintercept of the linear regression to the detection limit (e.g. Ihinger et al. 1994). This methods is not accurate enough and depends on the uncertainties of the regressed data, the leverage of the data for the higher concentrations being potentially unreasonable. Ideally, only the measurements of standards with F, S, and Cl concentrations lower than the expected background can give the detection limit (see Koga et al., 2003, for Cl (Fig. 2). These values are close to the zero intercept and considering the error on the y-intercept is as large as the value itself, the linear regression of the calibration curve is equivalent to forcing the regressin through zero. The slopes between forcing the linear regression through zero (red curve Fig. 2) and classic linear regression (blue curve Fig. 4) is identical within error. A detection limit of <1

Elastic Recoil Detection Analysis (ERDA)

ERDA is an absolute measurement independent from the two previous methods (EPMA and SIMS). Absolute because it consists of a shock between the nuclei of 2 atoms and the radii of the two nuclei (for example 19 F and 127 I) are known with great precision, and therefore the ERDA method do not require any standard to perform a measurement. ERDA has previously been used to measure hydrogen in geological materials (e.g. Mosbah et al., 1990;Bureau et al., 2009) The recoil masses are identified by means of a coincident measurement of the particle velocity and total energy. The recoil energy for 19 F is 3.5 MeV and that of 35 Cl is 5.3

MeV. Here a gas ionization chamber (GIC) instead of silicon detectors has been used

for energy measurements because silicon detectors suffer from considerable radiation damages. This standard-free method gives absolute F and Cl concentrations. The shape of the beam on the sample is a rectangle of 1 mm × 4 mm but only a small part of it was actually targeting the sample, the rest was hitting the surrounding indium. A classical ERDA graph displays a time of flight curve for each ion versus energy (Kottler et al. 2006). These curves are processed to extract a spectrum for each elements (Fig. 3a,b,c)

Results

There is a good general agreement on mafic standards for the measurements of F, S and Cl between EPMA and SIMS above a certain treshold of concentrations, >150 µg.g-1 for F, and >20 µg.g-1 for S and >20 µg.g-1 for Cl (Fig. 4). For F<150 µg.g -1 SIMS can measure differences in F concentrations with a precision better than 10%

relative when EPMA has a precision equal to the measured value (Fig. 4a). The performance of both EPMA and SIMS are in excellent agreement for Cl measurements down to 20 µg.g-1 (Fig. 4c). But for S measurements, SIMS can measure S concentrations below 10 µg.g-1 when EPMA will not measure resolvable difference in standards with S<10 µg.g-1 (Fig. 4b).

The glass standards measured here have reported value that can vary up to a factor of 10 for certain elements (ex: Cl in StHs6/80; (1982) and this has nothing to do with the quality of the analysis. In this respect, ATHO-G and StHs6/80-G appears to be heterogenous for F, S and Cl depending on the pieces you have. Also the 2error we report for our EPMA are 9 times out of 10 better than previously reported, demonstrating that the proposed settings for halogen measurements by EPMA are particularly well suited.

The ERDA results for F and Cl in ATHO-G, KE12 and KL2-G anchors independantly the calibration curves for F and Cl. We note that the ERDA values for F and Cl of the two SiO2-rich standards, are closer to the EPMA values than the SIMS values (Fig. 4a and 4c), and the ERDA measurement on the basalt standard KL2-G was difficult to assess because of the high detection limit of the ERDA.

Discussions

Precision and accuracy

The lowest concentrations we measured were samples GOR-128 and GOR-132 for F and S and sample StHs for Cl (Table 2). While EPMA measurements tend to level out around 10 µg.g-1 concentration for S (Fig. 4b), SIMS measurements are precise to µg.g-1 level for S and Cl (Fig. 4b andc). For F, S and Cl, SIMS measurements always display smaller error bars (Fig. 4a,b,c). Samples with concentration in S, Cl > 100 µg.g-1 are analyzed with similar precision with both methods. Measurements of F remains up to 5 times more precise with SIMS than EPMA on the basis of analytical precision based on counting statistics and for F concentration above 100 µg.g-1. Because many standard values are still tied to EPMA measured samples, it appears that the uncertainty of the slope and intercept (Fig. 2) contributes to the final uncertainty similar to EPMA values (Table 2). Therefore, it is strongly recommended to use SIMS when the interest of measurement is to detect variations of concentration among similar samples with a high precision, while EPMA can certainly provide a rapid, good assessment of trace volatile abundances above a certain threshold.

Matrix effect

It is particularly notable that some measure values by SIMS (reported for ATHO-G and KE12; Table 2) significantly differ from those of EPMA and ERDA. the EPMA data below detection limit. It appears that RSF is slightly negatively correlated against SiO2, consistent with a presence of matrix effect for the high SiO2 samples. However, considering the scatter of RSF values, the appearent negative correlation has only slight statistical significance. The data aquired here is not sufficient to decern the exact role of the "matrix effect". Because of such tendency, van den Bleeken and Koga (2015) concluded from a similar analysis that as a first order, one should be able to approximate the abundance of these element without correction.

Choices of calibration method

Reference mass: concentration analysis by SIMS requires a ratio of the element of interest (F, S, Cl here) over an element that constitute the matrix. For silicate glasses, it is commonly Si is chosen (Shimizu and Hart). 30Si is commonly selected for its low abundance permitting the use of electron multiplier detector. However, depending on the SIMS facility, different reference mass is used. For example, 28Si detected with Faladay cup can be used as the reference mass, as well as 18O-or 16O-. In general, emission of negative oxygen atom is approximately 10 times better than Si but this does not seems to result in significantly more stable signal. While it will require further study to assess the advantages and disadvantages regarding the choices of the reference mass, a comparison results from different SIMS labs concluded that it would not influence the measurement significantly (in the electronic supplement, van den Bleeken and Koga, 2015).

Calibration curves: a linear function that converts a SIMS intensity ratio to a concentration can be expressed in following two ways.

[F, S, Cl ppm] = Coef x (I(F, S, Cl) / I (Si) ) x [SiO2] + Intercept (eq. 1)

[F, S, Cl ppm] = Coef x (I(F, S, Cl) / I (Si) ) + Intercept (eq. 2)

where, brackets indicates concentration and I(x) indicates SIMS intensity (i.e. count rate) of mass x. Coef, and Intercept are constants determined by fitting the function using known concentration standards. Among SIMS measurements reported, these two equations were commonly used. The eq. 2 is sufficient for the measurement with a good match of sample and standard matrices (i.e. similar SiO2 content). In the current study, we adapted eq. 1, which corrects for variable SiO2 content (e.g. 50% basalt and 70% rhyolite). However, it should be noted that the eq. 1 does not correct for the matrix effect.

Recognising the weak correlations between SiO2 and RSF, we have explored a potential modification of the working curve function in an aim to optimize the accommodation of SiO2 viriation in silcate glass. Taking Cl as an example, the eq. 1 can be rearranged to show the relationship with RSF.

{[Cl] -[Intercept]}/{[SiO 2 ]}×k = RSF×(I Cl /I Si ) eq. (1')
Where k is a conversion factor for concentration ratio to atomic ratio, thus Coef = RDF/k. Inspecting Fig. 5, we decided to explore two functional forms relating RSF and SiO2.

RSF = a / [SiO2] + b (eq. 3) RSF = c × [SiO2] + d (eq. 4)
Substitutig eq. 3 or eq. 4 into equ. 1', the working calibration curve will be in following form.

[Cl] = ak(I Cl /I Si )+bk(I Cl /I Si )[SiO2] +Intercept (eq. 5)

[Cl] = dk(I Cl /I Si )[SiO2] + ck(I Cl /I Si )[SiO2] 2 + Intercept (eq. 6)

Table 3 shows the result of regression with above equations. For the regression, in addition to six calibration standards, three high Si samples are added T1g, ATHO, and KE12. Inspecting the results of the regression, eq. 6 consistently produced better fit, although slight, than eq. 5. On top of this,'ck' term is significantly smaller than 'dk' term in eq. 6. This suggest that the role of additional term in correcting the matrixdependent calibration is minor. This conclusion is again consistent with that of van den Bleeken and Koga (2015) in which authors concluded the use of eq. 1 is sufficient to determine trace halogen concentration in a wide range of silicate glasses. Lastly, recalculated concentration using eq. 6 is presented in Table 2, indicated as SIMS [eq. 6].

Applications to arc lava olivine-hosted melt inclusions

Subduction zones have generally volatile-rich magmas. range of H 2 O abundance (Fig. 6). The F/Cl is normalized to the average F/Cl of each arc, so that all constant F/Cl ratio gather around the unity value. This illustrates that while H 2 O can vary due to degassing prior to (or after) the entrapment of melt inclusions, pre-entrapment F/Cl values remain constant. The same conclusion can be drawn with F/Cl plotted against CO 2 , F, or Cl. This systematics demonstrate either 1)

F and Cl remain in melt during degassing, a conclusion reached for example by Bucholz et al., (2013) or 2) partitioning of F and Cl between fluid and magma is identical (Wu and Koga, 2013;Dalou et al., 2014). While we focus on the incompatible behaviors of Cl and F in basaltic melt, it should be noted that there exists a number of studies and experimental work concerning Cl and F behavior in evolved magmas, with specific application to volcanology and ore deposit formations (e.g. Webster, 1990Webster, , 1992;;Brenan, 1993;Giordano et al., 2004). Their results on halogen partitioning in fluid -evolved melt systems show that Cl and F are strongly partitioned into the fluid at crustal degassing conditions (Webster et al., 2018;Dolejs and Zajacz, 2018). But even if the halogens appear to degas during magmatic evolution in mid-to-shallow crust, Cl and F in primitive basalts, especially in melt inclusions, appear to retain the information of magma genesis (Koga et al. 2009;Bucholz et al., 2013;Rose-Koga et al., 2012, 2014) We can now use, indifferently EPMA or SIMS for F and Cl measurements, on a large SiO2 range covering most of the geological samples.

Conclusions

An intercalibration of F, Cl and S measurements between EPMA and SIMS is reported for 10 glass-standards. Both analytical methods are in excellent agreement for standards with concentrations in these volatiles elements above 150 µg.g-1 for F and above 20 µg.g-1 for S and Cl. However, SIMS has a lower detection limit and is preferable in the case of low concentration samples. The ERDA measurements independently confirms and anchors our data. This study revealed a shift between 1). negative slope for all the panels however due to scatter of measurements, the trend is not statistically significant. There are less number of RSF values as many of sulfur concentrations are below EPMA detection limit and there were no independent way to verify their concentrations. Koga et al., 2012;Ecuador: Le Voyer et al., 2008;Narvaez et al., 2018;Shasta: Le Voyer et al., 2010;Mariana: Shaw et al., 2012;Lesser Antilles: Bouvier et al., 2008, 2010;Vanuatu: Sorbadère et al., 2011).

Tables Table 1: F, S, Cl and SiO 2 measurements in 6 basalt glasses with the corresponding analytical methods and references. They are the glass-standards used for the calibration on the WHOI SIMS. Guggino and Hervig, 2010;[3] Diaz et al. 2006;[4] Straub & Layne 2003;[5] Thordasson et al. 1996;[6] Dixon et al. 1991;[7] Thornber et al., 2002;[8] Coombs et al. 2004;[9] DeHoog et al. 2001;[10] Hall et al. 2006;[11] Fisk & Kelley 2002;[12] Witter & Kuehner, 2004;[13] Streck & Wacaster 2006;[14] Oskarsson et al. 1982;[15] Palais and Sigurdsson, 1989;[16] Métrich & Rutherford, 1991;[17] Mosbah et al, 1991;[18] Marianelli et al. 1995;[19] Cioni et al. 1998. [20] Le Roux et al., 2006. <d.l. means below detection limit. Numbers in italic are considered below detection limit.

  or to intercalibrate with infrared spectroscopy measurements (e.g.Aubaud et al., 2009; Withers et al., 2012). The ERDA were made at ETH Zurich, in the Ion Beam Physics laboratory of the Paul Scherrer Institut. We used a primary ion beam of heavy ion 127 I at 12 MeV. This iodine beam was produced by EN-tandem accelerator via cesium (Cs) sputtering of AgI. For lower projectile energies Time of Flight-ERDA (ToF-ERDA) is a widely used technique. The analytical protocol is only briefly explained in the following, full details can be found in C.Kottler et al. 2006 (and reference therein). The beam hits the polished plane of the sample with a low angle and the scattered element of choice (F and Cl, here) are detected by the ToF-ERDA dectector at the fixed angle of 36º.

Fig. 4

 4 also shows that higher SiO2 glasses (e.g. ATHO and KE12) plot on the right side of the 1:1 line, outside of +/-20 % bound (a conservative external reproducibility range), indicating SIMS measurements are higher than EPMA and ERDA. While such offset is not present for mafic glasses which have similar SiO2 content as the calibration standards. This offset is present for measurements of F, S and Cl. This systematic disparity related to the composition of material analyzed is called matrix effect, in which the secondary ion emission is influenced by change either structual or compositional variation of the matrix. The relative sensitivity factor (RSF) describes a bias of an elemental ratio introduced by SIMS: RSF = (Ci/Cref)×(Iref/Ii), where Ci and Cref are the known atomic concentration of mass i and mass ref, respectively and I denotes the measured signal intensity. Essentially, the slope of the calibration function is a representative RSF of several calibration standards. It should be noted that RSF cannot distinguish bias of the signal of interest (e.g. I F , I S , and I Cl ) from the signal of reference (I Si ). Fig. 5 shows the value of RSF calculated for the samples of Fig. 4 as a function of SiO 2, excluding

  The magmas are produced by mantle wedge melting induced by slab-derived fluids. Lava erupting from arc these volcanoes are at least partially degassed. Olivine hosted-melt inclusions found in these lavas, are silicate droplets trapped in a host-mineral (olivine here) are less affected by degassing, and shielded from interaction with their surrounding in the magma chamber and during magma ascent. Especially for the halogens Cl and F, at pressure and temperature conditions of melt inclusion formation, they are less prone (1) to degassing (if at all) than H 2 O (e.g. Carroll and Webster, 1994; Métrich and Wallace, 2008), (2) to diffusing through the host-olivine (e.g. Bucholz et al. 2013; Lloyd et al. 2013; Le Voyer et al. 2014). Recent experimental results have determined F and Cl partition coefficients between melt and crystals (Dalou et al., 2014) and put forward that the large variation of F/Cl in arc melt inclusions resulted from the composition, the amount of slab agent and the degree of melting. Our recent melt inclusion data combined with litterature data show that although F/Cl ratios in arc melt inclusions can vary between 0.1 and 4 (see fig 5A in Dalou et al., 2014), within each sample suite, for each volcano, F/Cl is relatively constant, over a

  along with other lithophile trace elements (such as REE). The subduction input/output mass balance calculations show that nearly 100% of Cl coming in subduction is incorporated in arc magmatism, compared to only about 50% of F (e.g. Straub and Layne 2003; Wallace 2005).Therefore Cl and F are ideal tracers to identify the fractionation process between the slab and the flux originating from it, especially since they are scarcely present in the mantle (F reservoirs, see for exampleKoga and Rose-Koga, 2016; 2018). It is generally considered that Cl shows an affinity to volatile-rich fluid and F shows an affinity to silicate melt (e.g.Schilling et al. 1980). An experimental study on F fractionation between aqueous fluid and mineral at pressures and temperatures relevant to subduction zone settings, demonstrated for example the strong affinity of F for the silicate phase(Wu and Koga, 2013).Studies of halogens in magmatic products cover a wide range of applications. Studies involving F and Cl measurements (and their ratios with trace elements) on arc samples are able to decipher the transport vector (melt and/or fluid) from slab to surface (e.g.Rose-Koga et al., 2014). Also F and Cl measurements combined with Pb isotopes can bring new constraints on the source of OIB lavas (e.g.Peterson et al., 2014) or on the volatile contents of the mantle endmembers (e.g.Rose-Koga et al., 2017). Halogens combined with Sr isotopes can decipher between mantle source Clenrichment and brine assimilation (e.g.Reinhardt et al., 2018) Implications F and Cl measurments by EPMA and SIMS have a general good agreement on standards glasses available to the scientific community(MPI-DING, Jochum et al., 2006). These are the two most common insturment for in situ halogen measurements and finally there performance are analysed and compared. The F and Cl ERDA measurements on 3 standards anchors independantly the EPMA-SIMS comparison curves and gives absolute F and Cl concentrations for these standards. The ERDA results also highlight the fact that there is a matrix effect on SIMS measurements of F and Cl in high-SiO2 standard. We propose a new equation to correct this matrix effect in the SIMS F and Cl data of high-SiO2 samples.

  acidic and mafic glass-standard on our ERDA data, thus requiring (1) cautious in the choice of standard materials, and (2) separate SIMS calibrations with standards covering the SiO 2 range of the samples. An alternative is to use the equation 6 that we propose to account for this matrix effect on the SIMS measurements of F, S and Cl. In arc melt inclusions, F/Cl ratios of series of inclusion from single volcanoes, remain constant over a large range of H2O concentration, suggesting these halogen do not degass. On a broader point of view, halogens such as F and Cl (also Br and I), associated with radiogenic isotopes and trace element ratios, are promising new tracers of fluid and/or melt transport from their source regions and for degassing processes.Acknowledgement: EFR-K thank O. Sigmarsson for the Sletta sample. EFR-K and KTK acknowledge support from Region Auvergne Rhone Alpes (SCUSI program). This is Laboratory of Excellence ClerVolc contribution number xxx.

Figure caption Fig. 1 :

 caption1 Figure caption

Fig. 2 :

 2 Fig. 2: SIMS calibration curves for abundances of (a) fluorine, (b) chlorine, (c) sulfur

Fig. 3 :

 3 Fig. 3: Elastic recoil detection analysis (ERDA) spectra for oxygene 16 O (a), fluorine 19 F (b) and chloride 35 Cl (c). On the y-axis are reported the counts and on x-axis, the

Fig. 4 :

 4 Fig. 4: concentrations of F (a), S (b) and Cl (c) measured by SIMS versus that

Fig. 5 :

 5 Fig. 5: RSF (the relative sensitivity factor) is plotted against SiO2 concentration in

Fig. 6 :

 6 Fig. 6: F/Cl ratio normalized to the average versus H 2 O concentrations in wt%.

  Fig.1

Table

  this procedure during low hydrogen concentration measurements by SIMS). It was not a simple task to verify ppm-level abundance, and we adapted calibration without explicitly identifying zero point count rate (Table1).

	Some studies have used adapted "blank" material such as San Carlos olivine and
	synthetic forsterite (Hauri et al. 2002, Le Voyer et al. 2017). With what was available
	to us we calculated detection limits 48 µg.g-1 for F, 3 µg.g-1 for S and 19 µg.g-1 for

Table 2

 2 Borisova et al., 2010) and cautious must apply when choosing your standards to perform micro-analysis. For example it is clear from Table 2 that the ATHO-G piece that we have is very different than the piece measured by SIMS in Jochum et al, 2006, and much closer to the composition of that of Oskarsson et al.

). Nonetheless, overall we note that technical improvement of in-situ instruments make it possible to reach interlaboratory agreements. Our EPMA and SIMS measurements most of the time agree within error with the reported values published since 2006

(ex: Jochum et al., 2006)

, simply improving the precision in some cases. When they do not agree, we can invoke millimeter scale heterogeneity of the standards. They have been reported for trace elements in the ATHO-G rhyolite (MPI-DING;

Table 2 :

 2 Report of F, Cl, S and SiO 2 concentration measurements in 10 referenced material ( * ) and 3 other basaltic glasses (± is 2. Analytical methods and references are specified.

Table 3 :

 3 Result of error weighted regression of Eq. 5, and Eq. 6

Table 1 :

 1 F, S, Cl and SiO 2 measurements in 6 basalt glasses with the corresponding analytical methods and references. They are the glass-standards used for the calibration on the WHOI SIMS.

		F [ppm]	±	S ± [ppm]		Cl ± [ppm]	SiO 2 wt%	Mehod; Reference
	ALV519-4-1	90 30	950	95	45	23	48.9 EPMA; [1]
	ALV1654-3	997 150 1562	78	2914 146	56.7 EPMA; [1], [2]
	ALV1649-3	445 67	1640	82	1433	72	51.5 EPMA; [1], [2]
	GL03-D51-3	299 45	1126	57	182	18	49.5 EPMA; [1], [3]
	GL07-D52-5	431 65	1183	59	322	32	48.6 EPMA; [1]
	EN113-46D-2	124 37	877	88	45	23	49.5 EPMA; [1], [3]
	[1] Helo et al., 2012; [2] Michael & Cornell, 1998; [3] Simons et al. 2002.
	Relative analytical error for F concentration >200 ppm is 15% and F<200 ppm is 30%.
	Relative analytical error for S concentration > 1000 ppm is 5% , between 100 to 1000 ppm is
	10% and < 100 ppm is 50%.
	Relative analytical error for Cl concentration >400 ppm is 5%, between 50 and 400 ppm is 10%,
	and Cl< 50 ppm is 50%.
	Errors of these standards are assessed on the long term reproducibility.
	SiO2 was measured by EPMA

Table 2 :

 2 Report of F, Cl, S and SiO 2 concentration measurements in 10 referenced material ( * ) and 3 other basaltic glasses (± is 2. Analytical methods and references are specified.

					F [ppm]	±	S [ppm]	±	Cl [ppm]	±	SiO 2 wt%	Method; Reference
					70	18	1.2	0.12	7.5	1.4	51.4	SIMS; [1]
							100	100	110	33	EPMA; [1]
	ML3B-G	*		64;57;69	30;20;30	30	30	60	18	EPMA; [1] EPMA; SIMS; PIGE; [2]
					71	14	5.2	0.1	36	1	SIMS; [WHOI this study]
					61	11	5.1	0.2	32	2	SIMS; [eq. 6, this study]
					48	68	17	13	32	21	50.7	EPMA; [LMV this study]
					320	32	2.7	0.3	184	18	63.7	SIMS; [1]
							40	40	210	42	EPMA; [1]
									230	69	EPMA; [1]
	StHs6/80-G	*	139;122;155 2;2;2				EPMA; SIMS; PIGE; [2]
					<d.l.		4.3	0.1	27	0.6	SIMS; [WHOI this study]
					<d.l.		4.1	0.2	24	0.8	SIMS; [eq. 6, this study]
					28	35	19	1	21	11	EPMA; [LMV this study]
					321	32	2.6	0.3	113	14	58.6	SIMS; [1]
							30	30	100	50	EPMA; [1]
									130	65	EPMA; [1]
							0.92		119	LA-ICPMS; [3]
	T1-G	*					1.94			LA-ICPMS; [3]
					107;94;119	6;6;8				EPMA, SIMS, PIGE; [2]
					274	39	6.0	0.2	175	8	SIMS; [WHOI this study]
					207	29	5.7	0.2	135	14	SIMS; [eq. 6, this study]
					154	47	20	8	158	7	57.5	EPMA; [LMV this study]
					25	3	4.3	0.4	11.7	1	46.1	SIMS; [1]
							30	30	50	35	EPMA; [1]
	* GOR128-G	<d.l.		8.3	0.2	40 38	50 0.9	EPMA; [1] SIMS; [WHOI this study]
					<d.l.		8.4	0.3	33	1.7	SIMS; [eq. 6, this study]
					4	8	15	10	38	19	45.5	EPMA; [LMV this study]
					22	2	1.8	0.2	6.2	1	45.5	SIMS; [1]
							50	50	30	30	EPMA; [1]
	* GOR132-G	<d.l.		5.7	0.2	50 30	75 0.6	EPMA; [1] SIMS; [WHOI this study]
					<d.l.		5.8	0.3	27	1.0	SIMS; [eq. 6, this study]
					4	8	11	13	24	17	44.3	EPMA; [LMV this study]
					334	14				50.6	SIMS; [4]
	VG2 USNM * , aka 111240/52			1348 1365 1340 1305	124 58 160 135	291 316	104 38	EPMA; [5] EPMA; [5] EPMA; [6] EPMA; [7]
							1200	160	270	80	EPMA; [8]

Table 3 :

 3 Result of error weighted regression of Eq. 5, and Eq. 6 Intercept values are taken from the working curves of Fig.2. This choice was to reduce cases of erroneous fit coefficients. **RRMS : Reduced residual mean-square value, in which residual of fit values were normalized by the uncertainty of the standards, and the mean value of the sum of the square is reported. When RRMS > 1 indicates that fitted values on average plot outside of 1 sigma of the standard value.

		ak/dk ±	bk/ck ±	Intercept* ±	RRM**
	F (Eq. 5)	682 27	-5.0 0.5	-47	38	0.05
	F (Eq. 6)	17.8 0.8	-0.185 0.002	-47	38	0.04
	S (Eq. 5)	400 11	4.46 0.03	3.3	20	0.42
	S (Eq. 6)	19.8 0.6 -0.1466 0.0001	3.3	20	0.23
	Cl (Eq. 5)	14.0 13.6	12.1 0.2	19	17	0.49
	Cl (Eq. 6)	12.7 1.5 -0.0057 0.0005	19	17	0.47

*


Vincent Delauzun

Beatrice Amigues

Silvia Spinelli

Anais Gaubert

Philippe Leone

Magali Grange

Laurent Gauthier

Alain Roussel

Extracellular vesicles as a new platform to study cell-surface membrane proteins

Keywords:

Membrane protein interaction studies using extracellular vesicles Extracellular vesicles as a tool for structure determination of membrane proteins

Introduction

Cell-surface membrane proteins (MPs) [START_REF] Vinothkumar | Structures of membrane proteins[END_REF] are involved in many biological processes such as, cell signalization [START_REF] Grecco | Signaling from the living plasma membrane[END_REF] or viral infection [START_REF] Laura De Armas-Rillo | Membrane dynamics associated with viral infection[END_REF], and play an essential role in regulation of the immune response [START_REF] Reuven | Regulation of innate immune responses by transmembrane interactions: Lessons from the TLR family[END_REF]. These proteins which act as transporters, channels or receptors are preferred targets for drug researches and immunotherapies which are currently under development in pharmaceutical companies [START_REF] Narni-Mancinelli | Complement factor P is a ligand for the natural killer cellactivating receptor NKp46[END_REF]. In this context, structure and interactions of these proteins with potential ligands such as therapeutic antibodies must be investigated.

Entire cell-surface MPs can be produced and purified by extracting the protein out of the membrane through the use of detergents [START_REF] Kotov | High-throughput stability screening for detergent-solubilized membrane proteins[END_REF]. In addition to being long and expensive regarding to optimization needs, it is known that detergents do not stabilize membrane domains as efficiently as natural lipids in membranes, often leading to a partial to complete loss of activity/stability during protein extraction and purification [START_REF] Matar-Merheb | Structuring detergents for extracting and stabilizing functional membrane proteins[END_REF]. Recent developments have made it possible to stabilize full-length MPs into artificial membranes such as nanodiscs or proteoliposomes [START_REF] Yokogawa | Nanodiscs for structural biology in a membranous environment[END_REF] [START_REF] Console | Reconstitution in Proteoliposomes of the Recombinant Human Riboflavin Transporter 2 (SLC52A2) Overexpressed in E. coli[END_REF]. But these systems do not reflect the biochemical complexity of a real biological membrane and do not abrogate the use of detergents. Indeed, MPs embedded in a biological membrane such as a cellular lipid bilayer or a viral envelope can adopt complex orientations and conformations due to steric constrains depending on protein density and protein clustering pattern [START_REF] Garcia-Parajo | Nanoclustering as a dominant feature of plasma membrane organization[END_REF]. An alternative strategy may be used in case of monotopic MPs. Extracellular part without transmembrane domains can be expressed as a soluble protein which allows easier production and purification [START_REF] Pavsic | Expression, crystallization and preliminary X-ray characterization of the human epithelial cell-adhesion molecule ectodomain[END_REF]. These truncated proteins can be used for structure determination by X-ray crystallography, singleparticle cryo-electron microscopy (EM) [START_REF] Shtykova | The dimeric ectodomain of the alkali-sensing insulin receptorrelated receptor (ectoIRR) has a drop-like shape[END_REF] or for immunization purposes [START_REF] Bakkouri | Universal Vaccine Based on Ectodomain of Matrix Protein 2 of Influenza A: Fc Receptors and Alveolar Macrophages Mediate Protection[END_REF]. One could notice that these soluble extracellular domains may display different biochemical characteristics compared to the original full-length molecules, making them a questionable study model [START_REF] Tran | Spatial Localization of the Ebola Virus Glycoprotein Mucin-Like Domain Determined by Cryo-Electron Tomography[END_REF].

All living cells release extracellular vesicles (EVs) in their environment. In animal species EVs are found in plasma, urine, milk, sweat, saliva or tears [START_REF] Harding | Exosomes: Looking back three decades and into the future[END_REF]. EVs can be classified into three sub-families: apoptotic bodies, exosomes and microvesicles according to their biogenesis [START_REF] Van Der Pol | Classification, Functions, and Clinical Relevance of Extracellular Vesicles[END_REF]. Apoptotic bodies are produced during the apoptotic process, when cytoskeleton breaks up and causes the membrane to bulge outward [START_REF] Caruso | Apoptotic cell-derived extracellular vesicles: More than just debris[END_REF]. Exosomes are generated through the endosomal pathway and accumulate inside multivesicular bodies before being release by exocytosis [START_REF] Takahashi | Exosomes maintain cellular homeostasis by excreting harmful DNA from cells[END_REF]. Microvesicles are formed by modification of the actin's network that drives budding the plasma membrane. This process is comparable to the mechanism involve for enveloped viruses exit of the cells [START_REF] Van Niel | Shedding light on the cell biology of extracellular vesicles[END_REF]. MPs found on microvesicles surface are sorted in the cytoplasm by the ESCRT machinery [START_REF] Christ | Cellular functions and molecular mechanisms of the ESCRT membrane-scission machinery[END_REF]. Microvesicles size is about few tens to few hundred nanometers and they enclose soluble proteins, DNA and miRNA [START_REF] Cvjetkovic | Detailed Analysis of Protein Topology of Extracellular Vesicles-Evidence of Unconventional Membrane Protein Orientation[END_REF]. It has been recently

shown that microvesicles can be used to display recombinant MPs on their surface for structure determination by cryo-electron tomography [START_REF] Zeev-Ben-Mordehai | Extracellular Vesicles: A Platform for the Structure Determination of Membrane Proteins by Cryo-EM[END_REF].

Here we describe a simple method to produce EVs covered by recombinant MPs of interest and their use for cell-surface MPs-ligands interaction analyses. Other EVs-based downstream applications are also discussed as new tools for cell-surface MPs studies.

Material and methods

Plasmid construction

Vector

pCAGGS-mcherry vector (Addgene plasmid #41583) [START_REF] Niwa | Efficient selection for high-expression transfectants with a novel eukaryotic vector[END_REF] was used to produce EVs. This vector was digested with EcoRI-HF (NEB #R3101S) and BglII (NEB #R0144S) restriction enzymes in order to remove the mcherry gene out of the vector. Linearized vector was then separated by agarose gel (1 % m/v) electrophoresis and the DNA extracted band was purified by using a spin column from a DNA gel extraction kit (Biobasic #EZ-10) according to manufacturer instructions.

Genes

Several genes coding for different cell-surface MPs have been cloned into the pCAGGS expression vector for this study. Zaire Ebola virus glycoprotein Δ934-1389 cDNA (Zebov GP) was ordered from Genscript company as a synthetic gene cloned into the pcDNA3.1 vector (Thermo Fisher #V79020). This cDNA codes for the envelope's glycoprotein without its mucin domain [START_REF] Martinez | Impact of Ebola mucin-like domain on antiglycoprotein antibody responses induced by Ebola virus-like particles[END_REF]. Human Herpes Simplex Virus 1 glycoprotein B cDNA (HSV1 gB) [START_REF] Zeev-Ben-Mordehai | Two distinct trimeric conformations of natively membrane-anchored full-length herpes simplex virus 1 glycoprotein B[END_REF] was synthetized by IDT Technologies company as a codon optimized (for mammalian expression system) gblock gene fragment flanked with 5' and 3' hybridation sequences. Homo sapiens 5' ecto-nucleotidase cDNA (CD73) was kindly gifted as an amplified PCR product ready to be cloned by our collaborators from InnatePharma company. This cDNA encodes for the cellsurface CD73 human enzyme [START_REF] Schuler | Human CD4+CD39+ regulatory T cells produce adenosine upon coexpression of surface CD73 or contact with CD73+ exosomes or CD73+ cells[END_REF].

Cloning

Zebov GP and CD73 cDNAs were amplified by PCR with the Q-5 High Fidelity DNA polymerase (NEB #M0491L) according to manufacturer instructions and PCR products were purified by agarose gel (1 % m/v) electrophoresis and the DNA extracted bands were purified using spin columns from a DNA gel extraction kit (Biobasic #EZ-10).

PCR products (Zebov GP and CD73) and gblock gene fragment (HSV1 gB) were introduced into the linearized pCAGGS vector by using the In-Fusion HD cloning kit (Takara #639649).

Plasmids were then amplified under ampicillin selection after transformation of E. coli RapidTrans TAM1 competent cells (Active Motif strain #11096).

Primers and hybridation sequences

In-Fusion cloning parameters

Genes Primers

Zebov GP Zebov GP 5'-CATTTTGGCAAAGAATTGCCACCATGGGTGTTACAGGAATATTG-3'

Transfection

1) Detach the HEK293 EBNA cells from a 100 % confluent T175 flask using 5 mL of 0.25 % trypsin-EDTA solution (Gibco #25200-056).

2) Split the cells into 2 new T175 flasks with 35 mL of fresh complete DMEM medium.

3) Dilute 60 µg of plasmid DNA in 5 mL of OptiMEM medium (Gibco #31985-047) and 150 µL of Lipofectamine 2000 (Invitrogen #52887) in also 5 mL of OptiMEM medium.

Mix solutions together and incubate 5 min at RT. 4) Dispatch 5 mL of transfection solution into each T175 and mix.

EVs enrichment

Collect EVs containing supernatant (50 mL) and launch immediately differential centrifugation steps [START_REF] Livshts | Isolation of exosomes by differential centrifugation: Theoretical analysis of a commonly used protocol[END_REF].

1) Centrifuge 10 min at 500 g at 4°C in order to eliminate residual cells. Keep supernatant.

2) Centrifuge 10 min at 3000 g at 4°C in order to eliminate bigger cellular contaminants.

Keep supernatant.

3) Centrifuge 30 min at 10 000 g at 4°C. Keep supernatant. 6) Resuspend carefully each EVs pellet with 50 µL of PBS (NaN3 0.02 %) and store at 4°C.

EVs purification by SEC

EVs were then purified by size exclusion chromatography using a HPLC system (Dionex Ultimate 3000 from Thermo Scientific). 100 µL of EVs (absorbance at 280 nm between 2 to 3) were loaded on a Superdex 200 increase 10/300 GL prepacked gel filtration column (GE Healthcare Life Sciences #28990944), which contains an agarose based-resin, eluted during 60 min with PBS (NaN3 0.02 %) with a flow rate at 0.6 ml/min with UV detection at 280 nm.

EVs-containing SEC fractions (100 µL) were collected and stored at 4°C for further analysis.

For Cryo-EM experiments, SEC fractions were pooled and concentrated via ultrafiltration by passing SEC fractions through Vivaspin-500 10 kDa MWCO (GE Healthcare Life Sciences Electrophoresis was performed at 100 V (constant) and bands observed after staining with a 0.12 % Coomassie blue solution (VWR, #443286M). For western blot analysis, 20 µL of EVs (Abs = 0.5) were loaded on SDS-PAGE gel and a pre-stained protein ladder was used (Euromedex #06P-0111). After electrophoresis, the gel was blotted onto PVDF membranes (Immobilon-P 0.45 µm #IPVH00010, Merck Millipore). After blotting, membranes were blocked in PBS -0.1 % Tween-20 -3% BSA for 1 hour at room temperature. Membranes were then incubated with serum from a previously immunized llama (serum dilution 1/2000)

for 1 h at room temperature. After two 10 minutes wash in PBS -0.1 % Tween-20 steps, membranes were incubated with a goat anti-llama-HRP conjugate antibody (dilution 1/5000) (Bethyl #A160-100P) for 1 h at room temperature and the wash steps were repeated. Finally, membranes were subjected to HRP-detecting substrate (Immobilon Crescendo #WBLUR0100, Merck Millipore) and imaged on Amersham Imager 680 System (GE Healthcare).

Mass spectrometry

Protein strips were cutted from the SDS-PAGE, digested with Trypsin and analyzed by Maldi-Tof/Tof (UltraFlexIII Bruker Daltonics). The peptides resulting from the digestion were compared to the list of theoretical enzymatic digestion peaks of the expected sequence. In parallel, a query of the Swissprot database by the Mascot software (Matrix Science) was carried out to detect contaminants.

Biolayer Interferometry

BLI experiments were performed using an Octet Red96 (FortéBio) with protein A-coated biosensor tips (Fortébio #18-0028 A mouse anti-CD73 antibody (IPH53, 5 µg/mL) or a non-relevant antibody (5 µg/mL) were loaded on protein A sensors for 1 min. The sensors were then rinsed in PBS buffer for 2.5 min to remove excess of antibody and then incubated with 200 µL (Abs280 = 0.5) of purified EVs expressing CD73 membrane protein for 4 min for the association step. The sensors were then dipped in PBS buffer for 8 min for the dissociation step. Measurements were analyzed with FortéBio Data analysis software version 7.1.

Negative staining EM

6 µL of SEC purified EVs (Abs280 = 0.2) were deposited onto a glow-discharged carboncoated grid (Ted Pella Formwar/Carbon, 300 mesh, Cu #01813-F) using a PELCO easiGlow Glow Discharge Cleaning System (Ted Pella Inc., USA). Current: 25 mA, time: 25 sec and incubated for one minute. Sample excess was blotted off with a filter paper, rinsed with two 20 μL drops of water and stained with 10 µL of 2 % uranyl acetate for 30 sec. Micrographs were recorded on a Veleta 2K x 2K CCD camera using a Tecnai T12 Spirit electron microscope (FEI Company) operated at 120 kV and a magnification of 110 000 X or 220 000 X.

Cryo-EM

4 µl of SEC purified EVs (Abs280 = 2) were deposited onto a glow-discharged perforated carbon-coated grid (Ted Pella LACEY Formvar/Carbon, 300 mesh, Cu #01883-F) using a PELCO easiGlow Glow Discharge Cleaning System (Ted Pella Inc., USA). Current: 25 mA, time:

25 sec and incubated for 5 sec. The excess liquid was backside blotted off with a filter paper (Whatman filter papers quality 5 #1005-055) for 3 sec, the grid was then quickly plunged into liquid ethane using a Leica EM-GP cryo-chamber. EM grids were stored in cryo-boxes under liquid nitrogen until use, then mounted in a Gatan 626 cryo-holder and transferred in a Tecnai F20-FEG electron microscope (FEI Company) operated at 200 kV. Images were recorded with an USC1000-SSCCD Gatan camera and magnification of 110 000 X.

Immunization

Llama immunization

A llama (Lama glama) was immunized with purified HSV1 gB EVs (Ardèche-lamas France). 1 mL of HSV1 gB EVs (Abs280 = 0.1) were injected subcutaneously five times at 1-week intervals using incomplete Freund's adjuvant. Blood samples were collected aseptically 50 days after the last boost and serum isolated [START_REF] Desmyter | Camelid nanobodies: Killing two birds with one stone[END_REF].

Serum ELISA

Pre-immune and immune sera were collected at day 0 and after 50 days of immunization.

100 µl of SEC purified HSV1 gB-EVs (Abs280 = 0.1) were adsorbed on Nunc-Sorp plates (Thermo Fisher #442404). The immune serum conversion was analyzed by ELISA, making serum dilution 1/250 or 1/500 in PBS and by using goat anti-llama-HRP conjugate (Bethyl #A160-100P) and TMB reagent (Sigma #T5525-50TAB) to develop the ELISA reaction.

Absorbances of wells were recorded at 450 nm on a Safire 2 microplate reader (Tecan).

Results and discussion

We used several cell-surface MPs that are currently under studies in our laboratory to develop and validate our production process (Fig. 1). The Ebola and Herpes glycoproteins which are involved in the viral life cycle [START_REF] Hunt | Filovirus entry: A novelty in the viral fusion world[END_REF][32] are present on the surface of the viral envelope which derived from portions of the host-cell membranes. The human CD73 enzyme [START_REF] Adzic | Unveiling the role of Ecto-5'-nucleotidase/CD73 in astrocyte migration by using pharmacological tools[END_REF] which convert AMP to adenosine and which is over-expressed on tumor cells was also studied as part of our collaboration with Innate Pharma company [START_REF] Perrot | Blocking Antibodies Targeting the CD39/CD73 Immunosuppressive Pathway Unleash Immune Responses in Combination Cancer Therapies[END_REF].

EVs production and purification

Ebola glycoprotein was chosen as the first target to validate EVs production process due to its large size and easy-recognized shape by EM [START_REF] Lee | Ebolavirus glycoprotein structure and mechanism of entry[END_REF]. EVs purity was monitored during all the purification process by Dynamic Light Scattering (DLS) analysis. Hydrodynamic radius distribution by volume was preferred to distribution by intensity in order to report the relative percentages of high and low molecular weight species in solution [START_REF] Stetefeld | Dynamic light scattering: a practical guide and applications in biomedical sciences[END_REF] [37].

To produce EVs, mammalian cells were transfected with the pCAGGS vector containing the gene corresponding to the glycoprotein. Four days after transfection, EVs were first separated from dead cells and cellular debris and concentrated by differential centrifugations following the enrichment protocol described in section 2.3.1. DLS measurements after the two ultracentrigation steps (fig. 2 A) showed that most of soluble proteins and small protein aggregates with size under 40 nm that probably came from cell culture medium have been eliminated. EVs were further purified by size exclusion chromatography (Fig. 2 B). Collected fractions were grouped into three pools numbered from 1 to 3 and corresponding respectively to the beginning (between 6.5 mL and 7.5 mL), the middle (between 7.5 mL and 8.5 mL) and the ending part (between 8.5 mL and 9.5 mL)

of the EVs elution peak and were analyzed by DLS (Fig. 2 C). Large protein aggregates and cellular membrane contaminants with a size from 200 nm to 500 nm were found in pool-1 while smaller debris with a size under 40 nm were eluted at the end of the peak in pool-3.

Proteins, DNA and RNA fragments that could remain after centrifugation steps [START_REF] Cvjetkovic | The influence of rotor type and centrifugation time on the yield and purity of extracellular vesicles[END_REF] have been also separated and eluted later during SEC purification (between 14 mL and 20 mL).

DLS analyses of Pool-2 showed hydrodynamic radius distribution ranging from 25 nm to more than 100 nm with a Z-average of about 65 nm. This pool was further analyzed by SEC-MALS, EM, SDS-PAGE and western blot.

EVs characterization

Molecular mass, size and stability of EVs

Purified EVs from pool-2 have been further characterized in terms of physical properties, stability and biochemical composition. Zebov GP-EVs concentration in pool-2 has been first evaluated by UV absorbance measurement at 280 nm. We found a fairly good correlation rate between the absorbance values of different pool-2 samples and the number of EVs seen on negative staining EM grids. Therefore, we decided to take the UV absorbance measurement as a way to estimate the quantity of EVs in the samples. EVs from pool-2 were then analyzed by size-exclusion chromatography (SEC) coupled with multi-angle light scattering (MALS) detector and refractive index (RI) detector for the determination of molecular mass and size distribution (radius of gyration). SEC-MALS analysis of EVs (Fig. 3 A) revealed a molecular mass ranging from 80.10 6 Da to 250.10 6 Da with a value of around 150.10 6 Da at the middle of the peak. Radii of gyration of EVs were comprised between 50 nm and more than 100 nm with a value around 65 nm at the middle of the peak. These results were consistent with the size measurements performed by DLS during the previous purification steps. Interestingly, EVs proved to be highly stable at 4°C for months according to analysis by DLS and to observation by EM of stored samples. Indeed, DLS profiles of a sample taken during the purification step and after storage for 6 months at 4°C were clearly identical (Fig. 3 B). This result was important to validate the use of EVs as a membrane protein storage solution, which allows to always have samples ready for their use for downstream applications.

Electron microscopy of EVs

EM images of EVs from pool-2 have shown high level of EVs purity and validate our purification process. Size distribution of EVs previously investigated by DLS and SEC-MALS could also be confirmed by EM (Fig. 4 A). Typical cup-shaped of EVs were visualized by negative staining EM as expected. Furthermore, 90 % of the EVs present on the EM grid were decorated with a MP. We were able to observe that the protein displayed all around the surface of the Zebov GP-EVs had the characteristic shape and size of the 10 nm height Ebola glycoprotein spike [START_REF] Beniac | Structure of the Ebola virus glycoprotein spike within the virion envelope at 11 Å resolution[END_REF]. We were also able to observe EVs in their native state directly frozen in solution by cryo-electron microscopy (Fig. 4 B). EVs appeared as expected with shape of perfect spheres and decorated with a protein that were displayed and regularly anchored on the EV membrane. The number of over-expressed Zebov GP trimers per EV (radius size around 50 nm) was estimated between 150 to 200.

Zebov GP identification

The presence of Zebov GP in EVs from pool-2 was then investigated by SDS-PAGE (Fig 5 A).

Zebov GP is synthetized as single-chain precursor and transferred into the lumen of the endoplasmic reticulum where it forms trimers [START_REF] Beniac | Structure of the Ebola virus glycoprotein spike within the virion envelope at 11 Å resolution[END_REF]. Then it is cleaved by furin enzyme into the receptor-binding GP1 and the fusion-mediating GP2 subunits and are incorporated into virions membrane to initiate infection. GP1 (which contains a mucin-like domain) and GP2

remained linked together by an inter chain disulfide bridge [START_REF] Weissenhorn | Crystal structure of the Ebola virus membrane fusion subunit, GP2, from the envelope glycoprotein ectodomain[END_REF]. GP1 possesses a highly glycosylated mucin-like domain important for promoting viral infection. This domain has been removed in the present Zebov GP construct. GP1 and GP2 were identified by SDS-PAGE under reducing conditions. GP1 (around 60 kDa) and GP2 (around 25 kDa) appeared as characteristic smeared bands due to their high glycosylation states [START_REF] Wang | Mechanistic understanding of N-glycosylation in Ebola virus glycoprotein maturation and function[END_REF]. According to the SDS-PAGE gel, it appeared that Zebov GP represents a large part of the total mass of proteins contained in EVs. The presence of GP in EVs was then confirmed by western blot. We are engaged in a project that aims to generate neutralizing camelids antibodies (also named nanobodies) against Ebola virus. Our laboratory hosts a platform dedicated to the generation and selection of nanobodies against various antigens. As part of this service, a llama had already been immunized with the extracellular domain of the Zebov GP. The serum from this llama was used to confirm the presence of GP1 and GP2 in EVs by western blotting (Fig 5 B). Finally, an identification by peptide mass fingerprinting of the SDS-PAGE GP1 band was performed. The proteolytically generated peptides were investigated by determination of molecular masses. For protein identification, the experimentally obtained masses were compared with the theoretical peptide masses of proteins stored in databases by means of mass search programs [START_REF] Thiede | Peptide mass fingerprinting[END_REF]. In the total mass spectrum, 70 % of peptides were identified with a sequence coverage of GP1 at 40 %. This partial coverage could be explained by the presence of glycosylation sites in the uncovered sequences, increasing the experimentally obtained masses of these glycosylated peptides. We could therefore conclude with certainty that Zebov GP was well expressed on EVs. Thanks to all these results, the same purification process was performed to produce CD73-EVs and HSV1 gB-EVs.

EVs applications

It has been previously shown by Dr Zeev-Ben-Mordehai and colleagues from Oxford University, that EVs can be used for surface membrane protein structure determination by electron cryo-tomography [START_REF] Zeev-Ben-Mordehai | Extracellular Vesicles: A Platform for the Structure Determination of Membrane Proteins by Cryo-EM[END_REF]. Here we present two other applications that we investigated: the use of EVs for molecular interaction studies and their use for immunization for antibody generation.

Interaction studies

CD73 is over-expressed on tumor cells and leads to an abnormal increase in adenosine levels resulting in a down-regulation of the immune response. Innate Pharma company has recently developed an anti-CD73 antibody (IPH53) which blocks this enzyme an restore the immune system activity [START_REF] Perrot | Blocking Antibodies Targeting the CD39/CD73 Immunosuppressive Pathway Unleash Immune Responses in Combination Cancer Therapies[END_REF]. A human CD73 enzyme displayed on EVs (CD73-EVs) and IPH53

antibody were used to investigate the ability of using EVs for ligand interaction studies.

CD73-EVs were produced and purified as described above and firstly observed by EM (Fig. 6 A). CD73 proteins have been localized all around EVs with a size and shape (5 nm height by 13 nm width) consistent with protein structure predictions [START_REF] Knapp | Crystal structure of the human ecto-5ʹ-nucleotidase (CD73): Insights into the regulation of purinergic signaling[END_REF]. However, we noticed that a part of the purified EVs (around 30 %) did not display the CD73 protein unlike what we observed in the case of the Zebov GP protein for which more than 90% of the EVs were decorated. The total amount of CD73-EVs obtained was also 3 times less than the amount of Zebov GP-EVs obtained for a same production scale. This may due to lower transfection efficiency rate or to lower expression yield for this cell-surface MP construct. Thanks to molecular CD73-EV / antibody ratio optimizations we were able to fit conditions to get relevant EM images of the IPH53 binding on CD73-EVs. The EVs appeared clearly covered with an additional outer layer of IPH53 visible all around and on top of the EVs. (Fig 6 B).

Many free antibodies were also visualized showing the excess of antibodies used.

We further investigated the interaction between IPH53 and CD73-EVs by biolayer interferometry experiment (Fig. 7). The association of CD73-EVs on protein A biosensor preloaded with IPH53 was monitored, which confirmed the interaction. We were unable to estimate any equilibrium dissociation constant (KD) between IPH53 antibody and CD73

antigen due to the corpuscular behavior of the EVs and their heterogeneous protein composition. Nevertheless, BLI experiments could be used to rapidly screen a large number of antibody interactions against cell-surface MPs displayed on EVs.

Antibody generation

Herpes virus entry into cells and membrane fusion require viral glycoproteins. Glycoprotein B (gB) is one of the fusion proteins of HSV1 [START_REF] Maurer | The structure of herpesvirus fusion glycoprotein B-bilayer complex reveals the protein-membrane and lateral protein-protein interaction[END_REF]. To investigate that EVs can be used for immunization purposes and antibody generation, HSV1 gB-EVs were produced and purified as describe previously and injected into a llama. Due to the high immunogenicity of EVs recently reported [START_REF] Wahlund | Exosomes from antigen-pulsed dendritic cells induce stronger antigenspecific immune responses than microvesicles in vivo[END_REF][46], we decided to injected to the llama 20-fold less gB protein (around 250 µg) than the amount of a soluble protein required for a standard immunization.

Blood from llama was collected 50 days after the last injection and serum isolated. The immune response of the serum against the gB protein was evaluated by ELISA using HSV1 gB-EVs and compared to a pre-immune serum response. Absorbance values of wells were two times higher when serum from the immunized llama was used which confirmed antibody generation against the gB protein (Fig 8 A). The positive immune response of the serum against the gB protein (around 110 kDa) was also confirmed by the presence of the band at the right size on western blot revealed using the serum after immunization (Fig 8 B).

Another band around 80 kDa specific to HSV1 gB-EVs probably corresponding to a truncated form of gB protein was also observed.

Conclusion

We have developed a simple and fast protocol for the production of EVs coated with a cellsurface MP ready to use for downstream applications (Fig. 9). Due to their high stability, EVs

give us a unique opportunity to store MPs in a biological environment. EVs can be used to investigate interaction between cell-surface MPs and potential ligands without the need for membrane protein extraction with detergent. Functional assays like enzymatic activity measurements can be performed directly on EVs. They can be also used as a powerful immunogenic antigen for antibody generation against MP extracellular part. Finally, due to their particular physico-chemical properties such as their size and composition, EVs constitute advantageous tools for structural studies by cryo-electron tomography of cellsurface MPs as close as possible to their natural state. Biolayer interferometry analysis of IPH53 binding to CD73-EVs (black line) using protein A biosensors. A non-relevant antibody was used as negative control (red line). Experiments were divided into 4 different phases: the baseline acquisition, the loading of antibodies on the sensor, another baseline acquisition corresponding to a washing step of the sensors, the association and the dissociation phase. EVs can be used for several downstream applications: biophysical studies (e.g. interaction studies with potential ligands) or functional studies (e.g. enzymatic activity studies), structure resolution (e.g. by cryo-electron tomography) or immunization purposes (e.g. llama immunization for nanobodies generation). Functional studies

4 )

 4 Centrifuge 3 h at 110 000 g at 4°C to pellet EVs in 2 ultracentrifuge tubes (Beckman coulter #355616) with fixed angle rotor 50.2Ti on Optima LE-80k Ultracentrifuge from Beckman coulter (UC-1). Discard supernatant. 5) Wash EVs pellets carefully with 25 mL of PBS (Gibco #14190-136) and centrifuge one more time 3 h at 110 000 g at 4°C (UC-2). Discard supernatant.

# 28932225 ) 2 . 4 . 2

 28932225242 according to the manufacturer's instructions. The relative protein absorbance of each EV samples was measured at 280 nm by NanoDrop (Thermo Scientific).Important considerations:-Do not use any HPLC column pre-filter -Do not use higher Vivaspin membrane cut-off[START_REF] Benedikter | Ultrafiltration combined with size exclusion chromatography efficiently isolates extracellular vesicles from cell culture media for compositional and functional studies[END_REF] 70 µL of EVs (absorbance of fractions at 280 nm beetween 0.1 to 1) were analyzed by Dynamic Light Scattering (DLS) by using the Zetasizer Malvern Nano-S instrument. Hydrodynamic size distributions were measured in duplicate at 25°C during 40-80 sec with a count rate between 200-600 kcps in a 3 mm light path quartz cuvette (Hellma #105251005QS). The number of replicates conducted in one round was adapted automatically in the range of 10-20 measurements. The initial material Refractive Index (R.I.) was 1.45, while dispersant (PBS) properties were R.I. = 1.33 and viscosity = 1.02 cP. Multi-Angle Light Scattering 50 µL of SEC purified EVs (Abs280 = 0.3) were analyzed by SEC-MALS on a Superdex 200 increase 10/300 GL coupled with a multi-angle light scattering device (Dawn 8, Wyatt Technology) and an Optilab TrEX differential refractive index detector (Optilab TrEX, WyattTechnology) operated with a HPLC system (Thermoscientific). Column was first equilibrated with PBS (NaN3 0.02 %) for several hours, until the base line of the Optilab TrEX signal was stable. The light scattering and concentration data were analyzed in ASTRA to determine molecular weight and size distribution. Inter-detector band broadening and alignment were applied using the algorithms in ASTRA 7 (Wyatt Technology, USA) using BSA protein standard according to manufacturer-recommended methods. The dn/dc used was 0.185 mL/g since phosphate buffer was used for SEC. The temperature controlled-autosampler was kept at RT.2.4.3 SDS-PAGE / Westernblot50 µL of SEC purified EVs (Abs280 = 1.4) were mix with DTT (50 mM), and 1X NuPage LDS sample buffer (Life Technologies #NP0008). The samples were preheated at 80°C for 5 min and 20 µL, 10 µL or 5 µL were loaded on a 4-20% Tris Glycine polyacrylamide gel (Nusep #NG31-420). A molecular-weight size maker was used (Euromedex #06U-0511).

Figure 1 :Figure 2 :

 12 Figure 1: EVs production workflow Figure 2: EVs purification analyses A) Hydrodynamic radius distribution of samples analyzed by dynamic light scattering (DLS)after each purification step: the first ultracentrifugation (UC-1, circle), the second ultracentrifugation (UC-2, square) and the size exclusion chromatography (SEC, triangle). B) SEC from the UC-2 sample: pool-1 (6.5 mL-7.5 mL, green box), pool-2 (7.5 mL-8.5 mL, black box), pool-3 (8.5 mL-9.5 mL, red box). C) Hydrodynamic radius distribution of each part of the SEC elution peak (pool-1: green line, pool-2: black line and pool-3: red line) analyzed by DLS.

Figure 3 :

 3 Figure 3: SEC-MALS and DLS analyses of EVs.A) Molecular mass (blue) and radius of gyration (orange) of Zebov GP-EVs from pool-2 analyzed by SEC-MALS. B) EVs size distribution monitored by DLS of a UC-2 sample (full) and after a six-months storage period at 4°C (UC-2 / 6 months, cross).

Figure 4 :

 4 Figure 4: Electron microscopy (EM) of EVs. A) Negative stain EM image (left panel) and close-up view (right panel) of purified Zebov GP-EVs (magnification 110 000 X). Zebov GP proteins (white arrows) are displayed all around EVs. B) Cryo-EM image (left panel) and close-up view (right panel) of purified Zebov GP-EVs (magnification 110 000 X). Zebov GP proteins (black arrows) are displayed all around EVs.

Figure 5 :

 5 Figure 5: Zebov GP identification. A) 20 µL, 10 µL or 5 µL of Zebov GP-EVs (Abs280 = 1.4) from pool-2 analyzed by SDS-PAGE and revealed by Coomassie blue staining. B) 20 µL (Abs280 = 0.5) of different EVs from pool-2 (lane 1: HSV1 GPB-EVs, lane 2: CD73-EVs, lane 3: Zebov GP-EVs) were analyzed by western blot and revealed with a llama serum.

Figure 6 :

 6 Figure 6: Electron microscopy (EM) of CD73-EVs with IPH53 antibody.A) Negative stain EM image of purified CD73-EVs (left panel) and close-up view (right panel) of CD73 protein (white arrows). The upper vesicle is decorated with CD73 while the lower one is not (magnification 220 000 X). B) Negative stain EM image of a purified CD73-EVs (left panel) covered with IPH53 antibody (black arrows) and close-up view (right panel). Free IPH53 antibody can be also observed (black boxes).

Figure 7 :

 7 Figure 7: CD73-EVs and IPH53 interaction analysis by biolayer interferometry (BLI).

Figure 8 :

 8 Figure 8: Llama immunization with HSV1 gB-EVs

Figure 9 :

 9 Figure 9: Downstream applications summarize

Figure 1 Cell

 1 Figure 1

-HEK293 EBNA cells used instead HEK293 allows better EVs recovery -Use of serum-free medium allows to eliminate bovine EVs 2.3 EVs Purification

	Important considerations:
	5) Incubate 5 h in a CO2 incubator at 37 °C with 10 % CO2.
	6) Replace serum-containing medium (DMEM) of each T175 flask with 25 mL of
	Freestyle293 serum-free medium with GlutaMAX™ (Gibco #12338-018).
	7) Incubate 96 h in a CO2 incubator at 37 °C with 10 % CO2 before collecting
	supernatant.

  and all experiments were temperature controlled at 25°C. Tips were hydrated in PBS buffer for 1 hour at room temperature prior experiments. Baseline was performed with tips dipped into PBS buffer in the absence of EVs. Experiments were controlled with the software Data Acquisition 8.2 (FortéBio).

). A black bottom 96-well microplate (Greiner Bio-One #655209) was filled with 200 μL of solution (PBS buffer, antibodies or EVs) and agitated at 1000 rpm,

Acknowledgements

We thank Maria Mate from the AFMB laboratory (Marseille, France) for biophysical characterizations support and Patrick Fourquet from the Centre de Recherche en Cancérologie de Marseille for mass spectrometry analyses. We thank Marion Decossas from the Institut de chimie et biologie des membranes et des nano-objets (Bordeaux, France) for cryo-electron microscopy time allocation. The structural biology platform of the AFMB laboratory (Marseilles, France) was supported by the French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INSB-05-01. This work was also supported by the Agence Nationale de la Recherche (ANR-14-EBOL-0001).