H.-W Hübers

S Schröder

K Rammelkamp

D S Vogt

O Gasnault

Contribution of a martian atmosphere to laser-induced breakdown spectroscopy (LIBS) data and testing its emission characteristics for normalization applications

Keywords: Mars surface, Spectroscopy, Experimental techniques, Mineralogy, Mars atmosphere

scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Laser-Induced Breakdown Spectroscopy (LIBS) permits rapid in-situ multi-elemental analysis and has been evaluated for applications in planetary science in a number of studies (e.g. [START_REF] Colao | Investigation of LIBS feasibility for in situ planetary exploration: An analysis on Martian rock analogues[END_REF][START_REF] Knight | Characterization of Laser-Induced Breakdown Spectroscopy[END_REF][START_REF] Sallé | Laser-induced breakdown spectroscopy for Mars surface analysis: Capabilities at stand-off distances and detection of chlorine and sulfur elements[END_REF]. The first extraterrestrially employed LIBS instrument is ChemCam on NASA's Mars Science Laboratory (MSL) [START_REF] Maurice | The ChemCam instrument suite on the Mars Science Laboratory (MSL) rover: Science objectives and mast unit description[END_REF][START_REF] Wiens | The ChemCam instrument suite on the Mars Science Laboratory (MSL) rover: Body unit and combined system tests[END_REF], which has been successfully analyzing materials in Gale crater since the rover's landing in August 2012 [START_REF] Maurice | ChemCam activities and discoveries during the nominal mission of the Mars Science Laboratory in Gale crater[END_REF]. NASA's upcoming Mars2020 mission with a similar rover will again apply LIBS in combination with other spectroscopy methods (Raman spectroscopy, time-resolved luminescence spectroscopy, and visible and infrared reflectance spectroscopy) with the follow-up SuperCam instrument [START_REF] Wiens | The SuperCam remote sensing instrument suite for the Mars 2020 rover: a preview[END_REF]. Moreover, LIBS has been proposed for missions to planetary bodies with thin or no atmospheres, which includes asteroids and comets, the martian moons, and Earth's moon (e.g. Lasue et al., 2012). Applying LIBS in high pressure environments such as on Venus (e.g. [START_REF] Arp | Feasibility of generating a useful laser-induced breakdown spectroscopy plasma on rocks at high pressure: Preliminary study for a Venus mission[END_REF] has been considered as well. The martian atmospheric pressure is close to ideal conditions for LIBS and leads to high signal intensities in comparison to Earth atmospheric pressure or higher and to vacuum [START_REF] Brennetot | Mars Analysis by Laser-Induced Breakdown Spectroscopy ( MALIS ): In uence of Mars Atmosphere on Plasma Emission and Study of Factors In uencing Plasma Emission with the use of Doehlert Designs[END_REF][START_REF] Knight | Characterization of Laser-Induced Breakdown Spectroscopy[END_REF][START_REF] Sallé | Laser-induced breakdown spectroscopy for space exploration applications: Influence of the ambient pressure on the calibration curves prepared from soil and clay samples[END_REF].

In the LIBS technique, radiation from a high-power pulsed laser is focused onto a sample from which material is ablated and a luminous plasma is produced (e.g., Cremers andRadziemski, 2006, Singh and[START_REF] Singh | Laser-Induced Breakdown Spectroscopy[END_REF]. Information on elemental composition is obtained from emission spectra of excited atoms and ions due to specific transitions of electrons and the associated characteristic spectral lines. Bremsstrahlung (free-free transition) and recombination (free-bound transition) of electrons in the plasma result in a superimposed continuous spectrum. Breakdown and plasma formation can occur in solid, liquid, and gaseous targets, but the breakdown of a gas requires a higher laser irradiance than the breakdown of a solid. Atmospheric conditions such as pressure and the composition of the ambient gas affect the plasma formation processes, its evolution and emission and, therefore, the emission lines in the LIBS spectra (e.g., [START_REF] Aguilera | Temperature and electron density distributions of laser-induced plasmas generated with an iron sample at different ambient gas pressures[END_REF][START_REF] Brennetot | Mars Analysis by Laser-Induced Breakdown Spectroscopy ( MALIS ): In uence of Mars Atmosphere on Plasma Emission and Study of Factors In uencing Plasma Emission with the use of Doehlert Designs[END_REF][START_REF] Cremers | Handbook of Laser-Induced Breakdown Spectroscopy[END_REF][START_REF] Effenberger | Effect of atmospheric conditions on LIBS spectra[END_REF][START_REF] Sallé | Laser-induced breakdown spectroscopy for space exploration applications: Influence of the ambient pressure on the calibration curves prepared from soil and clay samples[END_REF].

A typical ChemCam LIBS spectrum obtained from the martian surface does not only show the emission lines of the elements of the sample but due to a partial breakdown of the atmosphere also always contains emission of carbon and oxygen (e.g., [START_REF] Gasnault | Deciphering sample and atmospheric oxygen contents with ChemCam on Mars[END_REF][START_REF] Knight | Characterization of Laser-Induced Breakdown Spectroscopy[END_REF][START_REF] Rapin | Quantification of water content by laser induced breakdown spectroscopy on Mars[END_REF][START_REF] Schröder | Hydrogen detection with ChemCam at Gale crater[END_REF]. The atmosphere of Mars is mostly composed of CO 2 (96 %) with a slightly varying surface pressure of 7-9 mbar at the MSL landing site depending i.a. on the season [START_REF] Harri | Pressure observations by the curiosity rover: Initial results[END_REF]. For samples containing no carbon, the carbon in the LIBS spectra is provided only by the ambient martian atmosphere and can be used to investigate the atmosphere's breakdown and its contribution to the plasma emission. Oxygen on the other hand is present in most of the mineral phases found on Mars, e.g. in silicates or in OH groups, or for instance as absorbed or adsorbed water. The oxygen emission in the LIBS spectra is therefore due to both the oxygen in the sample and the contribution from the breakdown of the ambient atmosphere, making a calibration of oxygen in the samples generally difficult (Gasnault et al., 2012;[START_REF] Knight | Characterization of Laser-Induced Breakdown Spectroscopy[END_REF]. Carbon and oxygen emission lines are both used for normalization of hydrogen in ChemCam data, i.e. the hydrogen emission intensity is divided by the emission intensity of the carbon or oxygen emission line, as this was found to reduce the signal variation induced by variations in operational parameters like distance, pressure, focus quality [START_REF] Rapin | Quantification of water content by laser induced breakdown spectroscopy on Mars[END_REF][START_REF] Thomas | Characterization of Hydrogen in Basaltic Materials With Laser-Induced Breakdown Spectroscopy (LIBS) for Application to MSL ChemCam Data[END_REF]. However, the coupling of the laser to the sample surface, the ablation of the sample surface, the plasma formation and the plasma evolution all depend strongly on various parameters of the experiment, which in turn affects the composition of the plasma and the relative contribution of the atmosphere to the LIBS spectrum. Critical parameters include the irradiance on the sample's surface and geometric effects such as the incidence angle of the laser, the physical matrix of the sample such as its surface structure, the chemical matrix of the sample, and the ambient gas composition and pressure, (e.g., [START_REF] Cremers | Handbook of Laser-Induced Breakdown Spectroscopy[END_REF][START_REF] Kurniawan | Review of Laser-Induced Plasma, Its Mechanism, and Application to Quantitative Analysis of Hydrogen and Deuterium[END_REF][START_REF] Singh | Laser-Induced Breakdown Spectroscopy[END_REF]. Therefore, it is not clear if the emission intensity of an element of the atmosphere is appropriate to normalize an element in the sample. Disentangling the contributions of the elements of the martian CO 2 -dominated low-pressure atmosphere to the LIBS plasma of a particular solid sample and understanding their dependencies is important for martian data interpretation such as the search for organic matter [START_REF] Beck | Searching for Carbon on Mars with MSL ChemCam[END_REF] and ChemCam data processing applications such as the afore mentioned normalization [START_REF] Rapin | Hydration state of calcium sulfates in Gale crater, Mars: Identification of bassanite veins[END_REF][START_REF] Rapin | Quantification of water content by laser induced breakdown spectroscopy on Mars[END_REF][START_REF] Thomas | Characterization of hydrogen in basaltic materials with laser-induced breakdown spectroscopy[END_REF][START_REF] Ytsma | Effects of univariate and multivariate regression on the accuracy of hydrogen quantification with laser-induced breakdown spectroscopy[END_REF]. In particular, the effects of changing irradiance are of interest, as the laser spot diameter on the martian target is varying with the distance to the LIBS instrument's telescope on the rover (2-7 m), [START_REF] Maurice | The ChemCam instrument suite on the Mars Science Laboratory (MSL) rover: Science objectives and mast unit description[END_REF].

The objective of this work is to investigate the breakdown of the martian atmosphere and the emission of carbon and oxygen in the LIBS spectra with varying experimental parameters and samples.

Various laboratory tests in simulated martian atmospheric conditions were performed with pure silicon wafers, pressed silicon grains with different grain sizes, a transparent quartz plate, and several pure metals and metalloids. The influence of the laser energy on the sample was tested and time-resolved measurements were performed to investigate the emission from the elements of the atmosphere and from the samples. All results are discussed in the context of the suitability of the carbon and oxygen emission from a CO 2 -dominated low-pressure atmosphere as an internal standard for the normalization of particular emission lines in the LIBS spectra of martian targets.

Materials and Methods

Five different measurements were performed to investigate the effect of the laser energy (A), to study the temporal evolution of the emission lines (B), the effect of the target's grain size (C) and optical transparency (D), and the dependence on the sample's matrix (substrate) (E).

Samples

In order to allow for an easy discrimination of elements in the plasma coming from the atmosphere (mainly carbon and oxygen) and the elements from the sample matrix, simple and pure samples were chosen. For the measurements where the laser energy was varied (A) as well as for the measurements where the temporal evolution of the plasma emission and particular emission lines were studied (B), a pure silicon wafer was chosen. The effect of different grain sizes (C) was investigated with six samples with silicon grains of less than 25 µm, 45-63 µm, 90-125 µm, 125-180 µm, 212-250 µm, and 355-500 µm, respectively. These were pressed at 5000 kg into little copper containers with an inner diameter of 2.5 cm. A pure (99.999%), polished artificial single-crystal quartz (SiO 2 ) from ChemPUR with a thickness of 3 mm was used to study a transparent sample containing silicon (D). Five metal sputtering targets (aluminum, copper, iron, nickel, titanium) with a pureness of 99.6-99.99% also purchased from ChemPUR as well as a silicon and germanium wafer were used to study the variation of the breakdown and contribution of the martian atmosphere to the plasma on different substrate materials (E).

Experimental set-up

For this study the LIBS system at Deutsches Zentrum für Luft-und Raumfahrt (DLR) in Berlin was employed. The radiation from a Q-switched Nd:YAG laser operating at its fundamental wavelength of 1064 nm with 10 Hz repetition rate, 6 ns pulse duration, and a maximum output energy of 220 mJ was focused onto the sample surface. The energy of the laser can be varied by a set of gray filters. The laser beam was directed through a lens system with an effective focal length of 410 mm almost vertical (5° offset) onto the sample, resulting in a spot diameter of about 300 µm. A toroid mirror reflects the plasma emission onto the entrance slit of an echelle spectrometer (LTB Aryelle Butterfly) equipped with a timegated intensified charge-coupled device (ICCD) camera (Andor iStar) enabling a useful, continuous wavelength coverage from 190-374 nm (referred to as "UV-range") or 270-850 nm (referred to as "Vis-NIR-range" though this wavelength range covers UV radiation as well) with a spectral bin of 5-34 pm. A typical UV-range spectrum has about 25,000 bins while a Vis-NIR-range spectrum has 29,000 bins. A desktop computer controls the pulsed laser as well as the gating and amplification of the ICCD and the data acquisition. The spectrometer has an integrated Hg-lamp that is used for wavelength calibration in both spectral ranges.

The sample is placed in a dedicated simulation chamber where planetary conditions can be imitated by adjusting pressure, temperature, and atmospheric composition. Here, all measurements were performed at room temperature in a simulated martian atmosphere with an appropriate gas mixture composed of 95.55 vol.% CO 2 , 2.7 vol.% N 2 , 1.6 vol.% Ar, and 0.15 vol.% O 2 at 7 mbar. In order to prevent alteration and contamination of the simulated martian atmospheric conditions by ablated and sublimated material, a constant flow of the gas mixture through the chamber was kept during the measurement. The spectrometer is placed outside the simulation chamber and the light of the LIBS plasma passes through terrestrial atmosphere before being detected.

Experiments and measuring parameters

In the first experiment (A), the energy of the laser was set to seven different values, ranging from 4.4 mJ to 50 mJ on the silicon wafer inside the simulation chamber, i.e. after transitioning the optics and the window of the simulation chamber. For each spectrum the emission of 30 laser-induced plasmas was accumulated. Five positions were probed per adjusted laser energy by moving the sample after every 30 laser pulses. The spectra were recorded for both wavelength ranges in subsequent measurements. A delay time of 150 ns between the plasma initiation and the opening of the camera was chosen to gate out the dominant continuous emission typical for the early stages of the plasma evolution but to still detect ionic emission, which is also decaying fast. The plasma emission was then recorded for 10 µs each.

As our laboratory set-up provides a constant spot size with a diameter of approximately 300 µm on the sample for all laser energies, we report and display our results here as a function of the laser energy. However, it has to be mentioned that the parameter actually primarily controlling the emission line intensities (affecting laser ablation processes and therefore the quantity of ablated mass and plasma characteristics, e.g., [START_REF] Singh | Laser-Induced Breakdown Spectroscopy[END_REF] and which has to be considered for the martian LIBS data is the irradiance on the target, i.e. the laser energy divided by the laser pulse length and the laser spot area. The applied laser energies in our experiment ranging from 4.4 mJ to 50.7 mJ translate into an irradiance of 10 -120 MW/mm 2 on the target. The ChemCam laser has a maximum laser energy output of 35 mJ which is reduced to 55 % on the remote target after having passed all the optics and the telescope [START_REF] Maurice | The ChemCam instrument suite on the Mars Science Laboratory (MSL) rover: Science objectives and mast unit description[END_REF]. For ChemCam, the laser spot diameter is changing with distance to the target, increasing from 300 µm for a distance of 1.56 m (calibration targets on the rover's deck) to 550 µm at 7 m. Most ChemCam data is taken at distances of 2.4 to 3 m [START_REF] Maurice | ChemCam activities and discoveries during the nominal mission of the Mars Science Laboratory in Gale crater[END_REF] with an estimated average laser spot size of 350 µm which results in a maximum irradiance of approximately 33 MW/mm 2 . In a laboratory experiment to characterize the ChemCam performance a similar maximum irradiance of 35 MW/mm 2 was measured at ambient terrestrial conditions [START_REF] Maurice | The ChemCam instrument suite on the Mars Science Laboratory (MSL) rover: Science objectives and mast unit description[END_REF]. Therefore, in terms of irradiance, the first four data points of our laboratory data cover a range that is similar to the irradiance that ChemCam can use on Mars. Since ChemCam data beyond ~5 meters is rarely taken and analyzed with special care only, the first data point from our laboratory data is also of minor relevance.

In experiment (B), the temporal behavior of the emission is investigated. The delay time was set to 12 different values from 0 ns to 2 µs. The laser energy was set to 15 mJ on the sample's surface. For each spectrum the emission of 30 laser-induced plasmas was accumulated and five different positions on the silicon wafer were sampled. Each plasma emission was recorded with a quite long integration time of 200 µs to ensure that all of the plasma emission is captured until the very late stages, where only neutral emission and molecular emission contribute. Experiments (C), (D), and (E) were performed with 15 mJ on the sample's surface and the same timing parameters as in experiment (A): delay time of 150 ns and an integration time of 10 µs. Five positions were analyzed on each target accumulating the emission of 30 laser-induced plasmas.

Data processing

A dark spectrum, i.e. a spectrum measured from the same location and for the same integration time as the active LIBS spectrum, was recorded at each position and was subtracted from the corresponding LIBS spectrum to account for instrument noise and stray light.

Emission lines of interest were fitted with a Lorentzian function and underlying linear continuum. Superimposed emission lines, if present, were fitted simultaneously. A fit with Voigt profiles did not improve the fit quality suggesting that the dominant broadening mechanism is due to the Stark effect in accordance with e.g., Capitelli et al., 2003 and[START_REF] Cousin | Laser induced breakdown spectroscopy library for the Martian environment[END_REF]. A quadratic fit to the continuum was tested as well to better match the local form of the sensitivity function of one order of the echelle spectrometer but did not change the results either.

Results

LIBS spectra and general characteristics

As stated in section 2.2, there are two different wavelength ranges available for the LIBS measurements which cannot be used simultaneously. Typical LIBS spectra of the silicon wafer obtained in simulated martian atmospheric conditions are shown in Fig. 1 (A) and (B) for both spectral ranges. In the , carbon and silicon emission lines with good signal-to-noise ratio (SNR) of three different ionization states can be found, see Table 1. With a laser energy of 15 mJ, the most intense emission lines are Si(III) at 254.2 nm, several Si(I) lines between 250-253 nm, and Si(I) at 288.1 nm followed by C(III) at 229.7 nm and C(I) at 247.8 nm with similar intensities. These two carbon emission lines are shown for different laser energies in Fig. 1 (C) and (D). There are no sufficiently intense oxygen emission lines in the UV range that would allow us to study their behavior in these experiments.

In the Vis-NIR-range and for a laser energy of 15 mJ, the most intense emission lines are Si(II) at 385.6 nm, 413.4 nm, 505.6 nm, and 634.7 nm, see Fig. 1 (B). Emission lines of three different ionization states are available again for silicon (Table 1). There are three weak C(II) doublets with only low SNR available in the Vis-NIR, the most intense being two C(II) lines at 658 nm close to the H(I) emission Fig. °1 (B) and zoomed view in (E). However, in the Vis-NIR-range the oxygen emission can be investigated with several ionic emission lines such as O(II) at 441.5 nm and 441.7 nm, the neutral triplet O(I) at 777.2, 777.4, and 777.5 nm, and the neutral O(I) 844.6 nm emission (also a triplet but not resolved with our spectrometer). Due to the available emission lines in the two spectral ranges the behavior of the carbon emission was studied with UV-range data while the oxygen emission was studied using the Vis-NIR-range data. The Si(I) 288.1 nm emission is detected in both spectral ranges and provides the possibility for comparison.

Experiment (A): Laser energy variation

When increasing the laser energy on the sample from 4.4 mJ to 15 mJ, the total emission in both spectral ranges increase linearly, see Fig. 2. For higher energies up to 50 mJ a linear trend of the total emission is only obtained in the UV-range. As this data was taken with a delay time of 150 ns, most of the continuous emission resulting from bremsstrahlung and recombination of the electrons has already decayed and mainly ionic and atomic emission is contributing to the total emission. Several emission lines of carbon and silicon with different ionization degrees in the UV-range are shown in Fig. 3. A logarithmic scale was used for the plots to display together the emission lines with very different intensities. The trends described in the following paragraphs were obtained from fits in linear space that are not shown here. The emission lines show different growth rates with increasing laser energy. The two C(II) emission lines studied here (283.7 nm and 283.8 nm) increase linearly within the tested energy range. The neutral C(I) emission at 247.8 nm is increasing non-linearly and shows a trend flattening towards higher laser energies, while the C(III) emission line at 229.7 nm becomes more and more steep. The intensity of C(III) is similar to the C(I) emission for a laser energy of 15 mJ while less intense for smaller energies and more intense for higher energies. For the silicon emission lines, similar trends are observed, see Fig. 3, bottom. The two Si(II) lines at 290.4 nm and 290.6 nm as well as the Si(I) emissions at 251.6 nm and 288.1 nm show nearly linear trends with laser energy. Emission of higher ionized atoms, here Si(III) at 254.2 nm and 308.6 nm, show a steeper trend for the lower energies which becomes less steep for higher laser energies. This can be explained by an increasing ionization ratio in the plasma with increasing laser energy.

In Fig. 4, top, a selection of the best observable emission lines of carbon and oxygen from the martian atmosphere together with the H(I) emission at 656.3 nm are shown for the Vis-NIR range. The hydrogen is probably mostly due to adsorbed water on the Si-wafer and to a lesser extent to the humidity in the simulation chamber. The observed oxygen emission results mostly from the contribution of the CO 2 -dominated atmosphere but possibly also to a much smaller extent from the water molecules. Since the three oxygen emission lines O(I) 777.2, 777.4, 777.5 nm show very similar trends, for the sake of clarity only the one at 777.2 nm is shown in the plot. Silicon emission lines with three different ionization degrees are shown in Fig. 4, bottom. There are many more intense Si(II) and Si(III) emission lines available, which have been fitted and checked for their trends with energy. As the same species show very similar trends only two per ionization degree are presented in the plot. In general, the intensities of the Vis-NIR range emission lines also show smooth trends with energy but with the exception of measurement #5 with a laser energy of 22 mJ that is probably an outlier. As in the UV, the Si(I) at 288.1 nm shows a close to linear growth behavior with laser energy. The other emission lines of both, the atmosphere and the wafer, show also close to linear trends for the first four data points but flattening towards higher laser energies. As in the UV-range, the higher ionized particles emission show a steeper trend with laser energy for the smaller laser energies due to the increasing ionization ratio in the plasma which becomes less steep towards highest laser energies applied here.

Experiment (A) shows that the intensities of emission lines vary with laser energy and that emission lines of the same element and with the same ionization degree show a similar growth behavior. The growth behavior of the emission line intensity with laser energy depends considerably on the degree of ionization of the emitting atom. The data shows that a ratio of two emission lines with different degree of ionization cannot be assumed to be constant when the laser energy or irradiance vary. Even a ratio of two line intensities of the same element coming from the same source such as Si(I) and Si(II) from the Siwafer is changing. Therefore, a ratio such as H(I) to C(II) as proposed in Thomas et al., 2015 as a normalized hydrogen value in ChemCam LIBS data is also likely to change with varying irradiance, i.e. with distance of the martian target to the rover. To what extend this particular ratio is affected in the martian ChemCam data cannot be estimated with our laboratory data but would have to be tested with a copy of the ChemCam instrument. We remind here that an irradiance range much larger than the typically applied irradiance of ChemCam on Mars was tested in our laboratory.

In order to evaluate if the ratio of two neutral emission lines provides a reliable ratio that remains stable with varying laser energy, the ratios of the five most intense neutral silicon emission lines in the UV-range to the neutral carbon emission C(I) at 247.8 nm were calculated. These Si(I)/C(I) ratios are shown in Fig. 5 (top) as absolute values. All Si(I)/C(I) ratios are decreasing for the first four data points, i.e. laser energies of 4.4 mJ to 15 mJ and then slowly rising again with a minimum typically between 20-30 mJ, except for Si(I) at 251.9 nm where the ratio is monotonically decreasing with no minimum. Over the tested laser energy range, these ratios vary up to a factor of ~2 and are most sensitive to changes for laser energies below 20 mJ. Since there is no neutral carbon emission line available in the Vis-NIR range, the ratio of Si(I) at 288.1 nm, the only neutral silicon emission line available in the Vis-NIR range, and the most intense neutral oxygen emission line O(I) at 777.2 nm was calculated, see Fig. 5, bottom. Here, a similar trend can be seen where the ratio is decreasing with increasing laser energy until a minimum is reached close to 30 mJ. The Si(I)/O(I) ratio varies up to a factor of 1.7 in our data and of 1.4 below 20 mJ. Similar results were obtained when using the sum of the oxygen triplet at 777 nm.

In comparison to Si(I), the ratio of the H(I) emission at 656.3 nm to O(I) shows a different trend and is increasing with laser energy with a weak maximum at 20-30 mJ (H(I)/O(I)=0.15 at 22 mJ). The sample includes no hydrogen and the hydrogen emission in the plasma is due to surface water or water vapor in the simulation chamber. Due to the big difference in intensity of the rather weak hydrogen and the intense oxygen emission line, the H(I)/O(I) ratio is quite small; the smallest ratio that was calculated in this study. Due to this small ratio and its variations between consecutive measurements, a typical H(I)/O(I) trend could be hidden in the data. Moreover, our obtained trend might not be representative as it requires less energy to dissociate H from surface water than ablate and vaporize it when bound in a solid sample matrix and the H(I) emission from surface water might show different characteristics with laser energy than from a hydrogen containing sample [START_REF] Kurniawan | Review of Laser-Induced Plasma, Its Mechanism, and Application to Quantitative Analysis of Hydrogen and Deuterium[END_REF]. We therefore advice against using the H(I)/O(I) of this data set for evaluating the general suitability of the H(I)/O(I) ratio. Still, this normalization of supposedly constant H(I) by O(I) seems to be quite stable, but more experiments with hydrogen containing samples will be necessary.

The data from both wavelength ranges also show that the ratio of a neutral emission line of the sample to a neutral emission line of the surrounding 7 mbar CO 2 -dominated atmosphere varies with laser energy. The highest variation for ratios of neutral silicon emission lines to neutral emission lines of carbon and oxygen from the atmosphere with laser energy was seen in our data for values that correspond to the irradiance that ChemCam is using on Mars (first four data points). However, the first data point is of minor importance since ChemCam is rarely taking data beyond 5 m as explained above. These results imply that the C(I) and O(I) emission of the martian atmosphere must be used carefully as an internal standard for normalization and only to compare samples for which the laser irradiance does not vary too much. Since the laser ablation and plasma forming processes and, therefore, the characteristics of the emission lines of the elements of the sample and of the atmosphere are complex and depend among others on the laser pulse properties, the application of our results to the specific case of ChemCam LIBS data would have to be tested with a copy of the ChemCam instrument itself in experimental conditions similar to the measurements on Mars.

Experiment (B): Emission line characteristics with time

The ChemCam instrument does not allow for time-resolved measurements and integrates approximately 3 ms [START_REF] Maurice | ChemCam activities and discoveries during the nominal mission of the Mars Science Laboratory in Gale crater[END_REF], which is quite longer than the total plasma lifetime of about 10 µs. However, the temporal evolution of the emission lines of the plasma was investigated here to learn about typical emission line characteristics of elements of the martian atmosphere and the sample with time. Finding differences or similarities in the temporal emission of the elements of the sample to those from the atmosphere helps evaluating if particular emission lines of the different origins can be compared to each other and if a ratio for normalization of e.g. hydrogen with carbon from the atmosphere seems reasonable. Furthermore, with the chosen integration time of 200 µs (gate time) in our experiments, the entire plasma emission until the very late stages is captured. The obtained emission line intensities are therefore an integrated signal and do not represent a momentary state of the plasma, for which a very small integration time would be necessary.

The total emission intensity recorded at different delay times after plasma initiation for the two available wavelength ranges is shown in Fig. 6. It is highest shortly after the plasma has formed and decays rapidly within the first several hundred nanoseconds, typical for plasmas induced with a Nd:YAG laser in martian atmospheric conditions as reported before (e.g., [START_REF] Schröder | Detection and identification of salts and frozen salt solutions combining laser-induced breakdown spectroscopy and multivariate analysis methods: A study for future martian exploration[END_REF]. In the Vis-NIRrange, the total intensity including the early phases of the plasma emission is higher than in the UV-range but is decreasing to lower values for delay times longer than about 300 ns. This indicates higher contribution of radiation due to fast decaying processes such as bremsstrahlung, recombination, and emission of ionized particles in the Vis-NIR range while the UV-range includes more long-lasting emission.

The UV-range emission line intensities of carbon from the Mars-analogue atmosphere are shown for different delay times in Fig. 7, top, and for the silicon emission of the silicon-wafer in Fig. 7, bottom. The emission of higher-level ionized species decreases faster while emission from neutral particles continues to the later stages in the cooling plasma. The trends are similar for emission lines of species with the same ionization degree. It can be seen that in our data the C(III) emission line at 229.7 nm is the most intense carbon emission line in the very early stages of the plasma, but disappears within the first 500 ns. The C(II) emission lines at 283.7 nm and 283.8 nm are less intense from the beginning and decay more slowly within approximately 700 ns. The neutral C(I) at 247.8 nm is emitting much longer and, in general, an intense C(I) emission line in the LIBS spectrum can be obtained with a long integration time (gate time). Similar trends mostly defined by the ionization degree of the species are also obtained for the silicon emission lines. Depending on their initial intensity, the emission of the ions disappears within the first several hundred nanoseconds and they do not contribute anymore for delay times longer than approximately 1.5 µs. Moreover, on this simple, mono-elemental sample, the decay rates of the emission lines seem not to depend on the initial intensity.

In Fig. 8, top, the emission line intensities of carbon and oxygen from the atmosphere in the Vis-NIR-range are shown together with the hydrogen emission for different delay times. Oxygen and carbon emissions are starting to decrease right after plasma formation like the silicion lines. The three oxygen lines of the triplet show again very similar trends so that only O(I) at 777.2 nm is shown representatively in the plot. Similar to the emission lines in the UV-range, the ionic emission decays more rapidly within several hundred nanoseconds while the emission of neutral oxygen is only slowly going down and contributing to the LIBS spectra also after 1 µs. The hydrogen emission, however, is rising at first and becomes most intense after approximately 250 ns. It then decreases similarly to the neutral oxygen emission. Hydrogen emission in LIBS spectra is known to show unusual behavior with exceptional processes taking place due to its low mass which might explain the temporal mismatch to the other emission lines (e.g., [START_REF] Kurniawan | Review of Laser-Induced Plasma, Its Mechanism, and Application to Quantitative Analysis of Hydrogen and Deuterium[END_REF]. To verify the temporal shift of the maximum intensity in hydrogen emission in comparison to the other species, time-resolved LIBS spectra in the Vis-NIR range of a silicon wafer in simulated martian atmospheric conditions were also recorded with a much shorter integration time of 500 ns (not shown here). The same trends for the atmospheric species were obtained with carbon and oxygen emission decreasing right after plasma formation and hydrogen increasing first and decreasing afterwards with a typical trend of a neutral species. However, the highest hydrogen intensity was obtained here slightly later than in the case of the 200 µs gate at a delay time of 500 ns. Silicon emission of particles with different ionization degree are shown for the Vis-NIR-range in Fig. 8, bottom. As seen in the UV-range, higher degree ionic emission such as the Si(III) decays within the first 1 µs while the neutral Si(I) emits beyond 1.5 µs.

The emission of neutral silicon normalized to the neutral emission of carbon and oxygen in the UV-range and in the Vis-NIR-range, respectively, is shown in Fig. 9. The Si(I) to C(I) ratio increases up to 40% within the first 500 ns and reaches typically similar values for longer delay times. The Si(I) 251.6 nm to C(I) ratio, however, shows a decreasing trend after 500 ns and after approximately 1.2 µs it is lower than right after plasma initiation. In the Vis-NIR-range, the Si(I) 288.1 nm to O(I) ratio is decreasing within the first 150 ns and then increasing, while H(I) to O(I) is increasing and reaches a maximum after about 800 ns. Both trends have been confirmed by the data with the 500 ns integration time. It is interesting to note the different trends with time of the neutral silicon emission at 288.1 nm divided by neutral carbon and in comparison by neutral oxygen emission. The differences in the temporal trends of these ratios could arise from the different origins of carbon and oxygen, where the former should only be provided by the CO 2 atmosphere while the latter is also coming from the surface water that leads likewise to the hydrogen emission in the LIBS spectra as discussed before. These results point to complex temporally and spatially varying distributions of the different particle species in the plasma that rely on initial geometries and reservoirs. From the increasing sample standard deviation it can also be seen that the variation in the measurements done at higher delay times after plasma initiation is increasing, suggesting that the later stages of the plasma emission are more prone to changes and are less repeatable, thus a lower precision, than the early phases.

These results do not directly contradict a normalization of sample emission lines with particular neutral and ionic atmospheric emission lines since a ratio of two integrated emission lines over time should not be affected in repeatable and similar plasmas. However, these results underline in particular the different temporal emission behaviors of particles of different ionization states and reveal their very different nature. Since varying irradiance and different samples lead to different ionization degrees, densities and distributions of the different particles in the plasma and can affect their emission times, these data are indirectly supporting the argument of not using a ratio of, e.g., a neutral particle to an ionized particle for normalization. Neutral and ionized species are moreover known to be spatially differently distributed in LIBS plasmas (e.g., [START_REF] Singh | Laser-Induced Breakdown Spectroscopy[END_REF], supporting this argument also from a geometric point of view. As has been shown, the ratio of the emission line intensities of two neutral particles also varies with time. For elements coming from different matrices such as from the ambient gas and the solid sample, the timing mismatch in emission line intensities could be due to a different location and therefore local temperature in the plasma. This could also explain the differences seen in temporal evolution of the neutral carbon and oxygen emission line intensities. This analysis also shows again that the hydrogen emission is special and behaves differently from other species in the plasma, emphasizing the need to very carefully explore and develop approaches for its calibration and quantitative analysis in the martian data.

SuperCam will allow for time-resolved LIBS measurements in the third wavelength range of 535-855 nm [START_REF] Wiens | The SuperCam remote sensing instrument suite for the Mars 2020 rover: a preview[END_REF] where hydrogen superimposed by ionic carbon and the strong oxygen emission at 777 nm can be detected. Time-resolved measurements, where a delay time of about 200 ns and a long integration time of several microseconds are applied, could allow for improved hydrogen detection as the superimposed carbon emission occurring in the early stages of plasma formation could be excluded.

Experiment (C): Effect of different grain sizes

It is known that the grain size can have an effect on the LIBS analysis of geologic samples (e.g., [START_REF] Anderson | The influence of multivariate analysis methods and target grain size on the accuracy of remote quantitative chemical analysis of rocks using laser induced breakdown spectroscopy[END_REF][START_REF] David | LIBS characterization of martian soil analogs: implications for the ChemCam analyses of aeolian sediments at Gale crater[END_REF][START_REF] Gottlieb | Impact of grain sizes on the quantitative concrete analysis using laser-induced breakdown spectroscopy[END_REF]. Here, the effect of different grain sizes on the emission of the elements of the atmosphere and of the sample was investigated since the breakdown thresholds for gases and solids are different and the thresholds are quite sensitive to the surface properties such as roughness [START_REF] Cremers | Handbook of Laser-Induced Breakdown Spectroscopy[END_REF]. Harder samples are expected to have higher ionic emission due to a higher speed of the shock wave that is typically exciting atoms in a secondary plasma at this pressure [START_REF] Kurniawan | Review of Laser-Induced Plasma, Its Mechanism, and Application to Quantitative Analysis of Hydrogen and Deuterium[END_REF]. These dependencies could differently affect the breakdown of the martian atmosphere and that of the sample on targets with varying grain sizes and closeness of packing in the pressed pellets.

The effect of different grain sizes was investigated with six samples with silicon grains of less than 25 µm, 45-63 µm, 90-125 µm, 125-180 µm, 212-250 µm, and 355-500 µm, respectively. For the samples with the small grain sizes, many grains were targeted at a time by the laser with a spot diameter of 300 µm while on the sample with biggest grain sizes only few grains were irradiated per position. When the laser spot is small in comparison to the grain size then the target appears consolidated and probability is high that only one grain is sampled per position. In this study we did not investigate silicon grains significantly larger than the laser spot size but also it would have not been possible to press grains of this size into a compacted and stable target. We did not do experiments on loose grains in this study.

The total intensities for both wavelength ranges are shown in Fig. 10 as a function of grain size. In comparison to the total intensity obtained on the silicon wafer with the same laser energy, the total intensities of both wavelength ranges from the pressed silicon grains are reduced to less than half. In our study, the total intensity of the plasma in the UV-range as well as in the Vis-NIR-range was most intense for the smallest grain sizes investigated here and shows a mostly decreasing trend towards grains similar in size to that of the laser spot. Most of that variation occurs between the finest grains (< 25 µm) and the next sieved portion (45-63 µm). For grains with the biggest size investigated in this study, which should be somewhat bigger than the laser spot, the total plasma intensity is slightly increasing. However, note that the variation in plasma intensity of measurements of the same sample is in the order of 10-20% and the trend with grain size is not very distinct for grains with the biggest size investigated in this study (> 63 µm), in particular not in the UV-range. The intensity of the neutral silicon, carbon, hydrogen, and oxygen emission lines in both wavelength ranges show similar vague trends but again variation in the data is high. In the Si(I)/C(I) and Si(I)/O(I) ratios, finally, no trend with grain size can be seen and variation between different measurements on the same sample dominate, see Fig. 11.

Experiment (D): Transparent sample

A polished piece of pure quartz (SiO 2 ) with a thickness of 3 mm was used to study a transparent sample containing silicon and to compare the results to the data obtained with the silicon wafer. Here, already the very first stages of ablation and plasma initiation are expected to differ a lot as the silicon wafer has a reflectance of the order of 30% for the infra-red wavelength of the LIBS laser while the quartz has a transmission of over 90%. The different initial processes due to the optical properties of the sample material could affect differently the breakdown of the sample material and that of the atmosphere. As the quartz also contains oxygen we are discussing the data here only with regard to a normalization of the silicon emission to the carbon emission in the UV-range.

The quartz sample was fixed at a distance of ~3 mm over a plate of germanium in order to prevent coupling to and contribution of a solid material from beneath the quartz target. Thus, laser light that was not deposited or reflected on the surface of the quartz encountered martian atmosphere again after passing through the quartz sample. The germanium plate was chosen here to be able to easily identify the origin of this emission in case it occurs. The laser was focused onto the quartz surface and emission of silicon, oxygen and carbon was detected. However, there were no germanium emission lines found in the LIBS spectra, suggesting that most of the laser pulse energy was deposited before.

Five different positions were analyzed on the quartz sample. Small craters formed where the laser was focused suggesting that most of the laser energy was deposited on the quartz surface. Also, fine cracks in the quartz appeared after the first laser shots, forming additional interfaces for the laser to couple to and facilitating energy deposition and plasma creation. Emission line intensities of the neutral silicon and carbon are shown together with their ratios in Fig. 12. Not surprisingly, the absolute intensities of neutral silicon are reduced in comparison to those obtained on the pure Si wafer and the Si grains but also the carbon emission is greatly reduced. While the silicon emission shows similar behavior for the five positions on the quartz, in particular the carbon emission on the 3 rd position stands out with higher intensity. The Si(I) to C(I) ratio is consequently reduced. This can also be seen to a lower extend on position #5. We attribute this differing emission as a result of the highly complex and unpredictable, chaotic breakdown on the transparent quartz sample where new cracks and interfaces are developing with every laser shot. In comparison to the Si(I) 288 nm to C(I) ratios obtained on the silicon wafer and the grains, the Si(I)/C(I) from the quartz sample is higher and also comes with a higher standard deviation of the five measurements due to the particularly complex nature of the partial breakdown of atmosphere and sample.

Although this data was obtained with a pure and perfect artificial quartz sample, the obtained results indicate that also on natural transparent targets unpredictable variation in the LIBS data can occur that affects the ratio of carbon from the atmosphere to the elements of the sample. Naturally occurring inclusions and impurities in geological samples could lead to even higher complexity in the breakdown and the relative carbon contribution to the LIBS plasma fluctuates.

Experiment (E): Varying substrate materials

Elements of the ambient atmosphere are known to show different emissions in LIBS plasmas depending on the substrate material (e.g., [START_REF] Ball | Hydrogen Leak Detection Using Laser-Induced Breakdown Spectroscopy[END_REF][START_REF] Senesi | Hydrogen Balmer α line behavior in Laser-Induced Breakdown Spectroscopy depth scans of Au, Cu, Mn, Pb targets in air[END_REF]. In order to study if and how the emission of the elements of the martian atmosphere vary on different materials, six other pure element samples were investigated in addition to silicon. The spectral range around the C(I) emission is shown in Fig. 13 for LIBS plasmas obtained on aluminum, copper, iron, germanium, nickel, silicon, and titanium in simulated martian atmospheric conditions. Different net intensities of the C(I) emission line are obtained depending on the substrate material and being highest on the iron target. It can be seen that iron has many emission lines in this wavelength region mostly coming from Fe(II) and in particular two emission lines just next to the C(I) emission. These were taken into account for the fitting, i.e. three emission lines were fitted simultaneously to the spectrum obtained from the iron sputter target. The net intensities of the C(III) and C(I) emission obtained on the different materials are shown together in Fig. 14 with the exception of C(III) on nickel, as here too many Ni emission lines were superimposing the ionic carbon emission. As can be seen from Fig. 14, the C(III) emission intensity is strongly affected by the material on which the plasma is produced, up to a factor ~6.7 difference between the Al and Fe substrates. But also the C(I) varies significantly depending on the sample, though the maximum difference in our experiment is reduced to ~1.6 between Fe and Ni substrates. On the other hand, neutral oxygen emission from the atmosphere varies much less on the different metal and metalloid substrates investigated here and seems more robust with a maximum difference of only ~1.2 between Cu and Ge substrates. This data also shows the problem for a normalization done with the total intensity as often applied for LIBS analysis as supposedly a proxy of the laser coupling efficiency with the sample: The total intensities of very different materials vary strongly in the UV-range as well as in the NIR-range, see Fig. 15. The differences in intensity are mostly due to different numbers of emission lines and the iron and titanium spectra of the UV-range feature maximum counts with their very many lines available there. In the Vis-NIR-range also titanium obtained maximum counts with a somewhat smaller value than in the UV-range. With the exception of aluminum, all samples analyzed here obtained higher total intensities in the UV-range than in the Vis-NIR-range. Also, the differences in total intensity in the UV-range and the Vis-NIR-range vary between the different samples and are moreover affected by the temporal gating parameters that were chosen for this measurement.

Having a closer look at the spectrum obtained on iron and identifying the emission lines of iron close to the carbon emission (cf., Table 2 and Fig. 16) it can be seen that the emission next to the C(I) actually comes from several Fe(II) transitions that are not resolved in our data. Moreover, it can be seen that there is another Fe(II) emission occurring at almost exactly the same wavelength than the C(I) emission. The Table 2 lists the positions and relative intensities of all ionic iron emission lines with a relative intensity index higher than 3000 in the proximity of the C(I) emission as obtained from the NIST database (https://dx.doi.org/10.18434/T4W30F). The wavelengths stated here are those for transmission in air as they are closer to the wavelengths that we observe with our setup where the plasma emission passes through Earth atmosphere before being detected. Martian atmosphere, however, leads to wavelengths more close to the values in vacuum [START_REF] Cousin | Laser induced breakdown spectroscopy library for the Martian environment[END_REF]. The relative intensity indices of one species usually cannot be directly compared with those of other species of the LIBS plasma (i.e., here the index of the neutral carbon emission) and it should be mentioned that the relative intensities of emission lines of LIBS plasmas in other ambient conditions such as in martian atmospheric conditions are known to deviate, cf. Cousin et al., 2011. Fig. 16 shows three LIBS spectra of pure iron all obtained at 6.5 mbar with different atmospheric compositions: Mars-analog gas mixture, terrestrial atmosphere, and helium. Moreover, the Fe(II) emissions with their relative intensity indices as listed in Table 2 are indicated by asterisks, shifted by -0.01 nm to better fit our laboratory data. The spectra obtained in Earth atmosphere and helium but at martian atmospheric low pressure show iron emission lines with intensities mostly in good accordance to the relative intensities from the NIST database. They show in particular the ionic iron emission at 247.86 nm that is one of the most intense in this spectral region. The spectrum obtained in martian atmospheric conditions is very similar to that obtained in Earth atmosphere at low pressure but features higher emission at 247.86 nm due to the additional emission of neutral carbon at this position.

These data show that there is a quite intense iron emission at the same position than the neutral carbon emission from the martian atmosphere. A martian target comprising iron therefore has additional emission superimposing the carbon emission. Extracting the precise intensity of C(I) by fitting this spectral region is hardly possible. Since most of the martian targets comprise iron there is iron emission in most of the spectral LIBS data. Extraction of the intensity of C(I) by fitting could only be done for very pure samples with no iron.

Discussion

The data of the five different experiments (A)-(E) conducted here show that normalizing an element of the sample with the emission of carbon or oxygen from the low-pressure CO 2 -dominated atmosphere can be problematic as this ratio depends on the irradiance of the exciting laser and changes with plasma life time. Additionally, the sample matrix affects the breakdown and amount of atmospheric compounds in the plasma. The ionization state of a species strongly affects the dependence of its emission lines on the irradiance and the temporal behavior of the emission. The ratio of a neutral emission line to an ionic line is therefore susceptible to changes and does not provide systematically a reliable and stable value in LIBS data, even more so when parameters such as the distance to the target and therefore irradiation are known to change between measurements. This is in accordance with the results obtained by [START_REF] Rapin | Quantification of water content by laser induced breakdown spectroscopy on Mars[END_REF], where the ratio of H(I) with C(II) was found to increase with distance from the rover. While the neutral species of the atmosphere and the sample show more similar behavior with irradiance and in temporal decay, the ratio of these emissions was still found to also change to different degrees in our experiments. The most crucial variation in our data was found in the range of irradiance that corresponds to that used by ChemCam on Mars. This will scale the reproducibility of the emission of an element of the sample scaled with that of an element of the martian atmosphere due to changes in irradiance that can result from variations in laser energy output (≤ 2% rms for ChemCam, [START_REF] Maurice | The ChemCam instrument suite on the Mars Science Laboratory (MSL) rover: Science objectives and mast unit description[END_REF] or due to different distances to the targets (also restricted in ChemCam collection, [START_REF] Maurice | ChemCam activities and discoveries during the nominal mission of the Mars Science Laboratory in Gale crater[END_REF].

Geometric effects and deviations of the exact focus position could also differently affect the contribution of the sample and the martian atmosphere to the plasma and, therefore, change the emission line intensities of the elements of the two origins in different ways. A defocused laser can lead to a higher amount of ablated mass due to a larger illuminated area on the sample but also the irradiance will be reduced at the sample's surface (Garcia et al., 2008[START_REF] Kurniawan | Review of Laser-Induced Plasma, Its Mechanism, and Application to Quantitative Analysis of Hydrogen and Deuterium[END_REF]. Similar to terrestrial applications, the martian atmosphere is likely to contribute to the plasma to a greater extent if the laser focus position is above the sample's surface than when the laser is focused below the surface. It will be part of a future study to investigate these effects in detail for a Mars-like ambient atmosphere. Moreover, due to their different origins, the elements of the atmosphere are differently distributed in the plasma than the elements of the sample (e.g., [START_REF] Aguilera | Spatial characterization of laser-induced plasmas by deconvolution of spatially resolved spectra[END_REF][START_REF] De Giacomo | Spatial distribution of hydrogen and other emitters in aluminum laser-induced plasma in air and consequences on spatially integrated Laser-Induced Breakdown Spectroscopy measurements[END_REF][START_REF] Kurniawan | Review of Laser-Induced Plasma, Its Mechanism, and Application to Quantitative Analysis of Hydrogen and Deuterium[END_REF] which is also indicated by our time-resolved data. The recorded plasma emission might therefore change and varying amounts of emissions from these distinct origins due to e.g. a changing field-of-view and/or incidence angle of the laser on the sample would be detected, which could change the emission line ratios in the obtained spectra. Spatially resolved spectra taken under martian atmospheric conditions will be helpful to understand the distribution of the elements of the sample and of the 7 mbar CO 2 -dominated atmosphere in the plasma. The spatial distributions of carbon, oxygen, and the exceptionally behaving hydrogen [START_REF] Kurniawan | Review of Laser-Induced Plasma, Its Mechanism, and Application to Quantitative Analysis of Hydrogen and Deuterium[END_REF] are of particular interest and will be addressed in a future study by means of time-resolved plasma imaging [START_REF] Vogt | Time-resolved spectral imaging of LIBS plasma at low pressures for the exploration of Solar System Bodies[END_REF]. The fact that ionized and neutral species are differently distributed in the plasma [START_REF] Singh | Laser-Induced Breakdown Spectroscopy[END_REF] adds to the list of arguments against comparing the emission lines of particles with different ionization degree also from a geometric perspective.

A big impact on the absolute carbon emission as seen from the experiments (D) and (E) comes also from the sample itself. The grain size has only a minor effect on the ratio of Si(I)/C(I) in our experiments and is of rather little importance in comparison to the intrinsic variability between different LIBS measurements on the compacted soil. However, the chemical composition and optical properties of the sample play a big role. The material characteristics such as its absorption coefficient affect the laser coupling and following processes for plasma formation and the relative contribution of the elements of the atmosphere to the plasma varies.

The targets in this study were chosen to allow for easy discrimination between elements of the martian atmosphere and those of a simple, mostly mono-elemental sample, and to reduce complex interferences. Though silicon is a major rock forming element, the pure silicon wafer might not be a very representative sample for an application where usually geological targets are probed. Our studied emission lines might also differ in their precise behavior from other elements and species of different samples. However, the data show that the ratio of a neutral emission line from the sample to neutral carbon and oxygen of the martian atmosphere is generally affected by different changing parameters of the experiment, suggesting that this can also occur to any other emission line ratio of elements of the two different origins. An individual univariate emission line ratio is moreover always depending on the specific transition properties of the emission lines such as the upper energy level (e.g., [START_REF] Hahn | Laser-induced breakdown spectroscopy (LIBS), part II: Review of instrumental and methodological approaches to material analysis and applications to different fields[END_REF]. Here, we studied several intense neutral silicon emission lines mostly available in the UV-range of our spectrometer without selecting particular transitions. Future experiments will be extended to include also geological samples. Moreover, special focus will be given to the hydrogen to carbon or oxygen ratio in particular, which was studied here only as a side effect due to the hydrogen present from adsorbed surface water. For the former ratio, a different set-up needs to be employed where the hydrogen emission in the NIR can be detected simultaneously with the carbon emission in the UV.

The last experiment showed that the neutral carbon emission in the UV is superimposed by iron emission to a considerable amount. Since the iron emission occurs at almost the same position, a reliable extraction of the intensity of the carbon emission by fitting the spectral region seems unfeasible when iron is present in the sample, neither with ChemCam resolution nor with the higher Echelle resolution of our set-up. In this regard, the oxygen emission seems better suited, as it is not superimposed by any other emission line. The three oxygen emission lines at 777 nm were found to show the same trends. This means that normalization could also be done with the sum of the triplet, which cannot be resolved in ChemCam data in any case. However, the big issue here is that oxygen is part of most minerals. To sample a martian target containing no oxygen is even less likely than to sample a target without iron. The samples investigated in experiment (E) did not contain oxygen in the sample matrix. However, the stability of the oxygen emission in our data from the different samples might be due to additional and similar amounts of oxygen coming also from the adsorbed water on the sample surfaces. While a possible out-leveling effect of the additional oxygen reservoir could be seen here as a positive thing and is consistent with the results obtained by [START_REF] Thomas | Characterization of Hydrogen in Basaltic Materials With Laser-Induced Breakdown Spectroscopy (LIBS) for Application to MSL ChemCam Data[END_REF], the question remains if the oxygen is a good general proxy for the plasma to be used for normalization. Applying such a normalization to a limited set of data for which the experimental conditions do not change too much in composition, laser energy, and distance would certainly help.

For all these reasons, a multivariate data processing approach is adopted for experiments like ChemCam for which all the experiment conditions cannot be systematically controlled with the hope that such a processing limits some of the effects reported here (Clegg et al., 2016 and references therein). However for some elements, such as minor species [START_REF] Ollila | Trace element geochemistry (Li, Ba, Sr, and Rb) using Curiosity's ChemCam: Early results for Gale crater from Bradbury Landing Site to Rocknest[END_REF][START_REF] Payré | Alkali trace elements in Gale crater, Mars, with ChemCam: Calibration update and geological implications[END_REF] or hydrogen, there is only one emission line that can be processed in ChemCam spectra and it is therefore important to understand the errors that can be introduced by the normalization as shown here and to evaluate the scale of these errors.

Summary and Conclusion

Different experiments were done in order to investigate the breakdown of a martian CO 2dominated low-pressure atmosphere and its contribution to the LIBS plasma emission when a simple solid sample is analyzed. The emission line characteristics were studied focusing on the dependencies on different parameters that are typically varying for targets analyzed by a robotic in-situ mission such as ChemCam on Mars.

Our results show that the ratio of two emission lines with different ionization degree strongly varies with laser energy. The trends of neutral carbon and oxygen emission from the atmosphere and neutral silicon emission from the sample show similar trends but the Si(I)/C(I) and Si(I)/O(I) ratios in our data were found to vary mostly for irradiances that correspond to martian ChemCam capabilities. The results indicate that in general the emission of an element of the atmosphere is of limited use for the normalization of the emission of an element of the sample matrix when the laser energy or irradiance is varying. Most of the ChemCam data are, however, taken in a delimited range of distances (< 5m), which may limit this effect.

The time-resolved measurements showed that in particular particles of different ionization states feature different temporal emission behaviors and that furthermore the ratios Si(I)/C(I) and Si(I)/O(I) vary with time when the plasma cools. This could be due to a different spatial distribution of the elements of the atmosphere to those of the sample and therefore different local temperatures of the different species in the plasma. ChemCam is integrating the total plasma emission and the different temporal decay rates should not affect the ratio of two emission lines. The results, however, indicate the complex spatially and temporally varying different distributions of the target's and the atmosphere's elements in the martian LIBS plasma. Future time-resolved LIBS plasma imaging in simulated martian atmospheric conditions will add more insight on this matter.

Our results show that the sample itself, its chemical and physical matrix, affects the breakdown and the amount of atmospheric compounds of the LIBS plasma. The grain size of the target was found to not strongly affect the ratio of neutral silicon from the sample to neutral carbon or oxygen from the atmosphere, as variations in the LIBS data from different spots on the sample are more dominant in our set of tests. Experiments done with a transparent quartz sample showed an even higher variability in the LIBS data of different spots on the sample. In particular, the ratio of Si(I)/C(I) varied strongly and was even higher than on the pure Si wafer. The amount of laser energy that is transferred into the breakdown of the atmosphere and into that of the sample randomly varies due to cracks arising from the laser-matter and plasma shockwave interactions. On natural transparent samples similar variation can occur, affecting the relative intensity of the carbon emission and impeding its use as a standard and robust signal for normalization. The absolute intensity of the carbon emission of the Mars analogue atmosphere from plasmas obtained on different pure metal and metalloid substrate materials was also found to vary. In comparison, the neutral oxygen emission from the 777 nm triplet was less affected by the sample material and seems to be a more robust proxy on different materials that comprise no oxygen.

The results indicate limitations of the suitability of the atmospheric neutral carbon emission at 247 nm as an internal standard. The oxygen at 777 nm emission shows similar dependencies with laser energy and in temporal evolution but was less affected by different sample materials. As it is also free from superposition of other elements it seems better suited as an internal standard proxy for normalization at least if there is no oxygen present in the sample or if its variation between the samples is known to be limited. 1: Overview of the emission lines of elements of the sample (Si) and elements of the atmosphere (C, O) in the two spectral ranges that are available with our LIBS set-up and were studied for this work. The titles "UV-range" and "Vis-NIR range" are names chosen merely to differentiate between the two spectral ranges even though the latter includes UV radiation as well.

Figure 1: High-resolution LIBS spectra of a Si-wafer obtained with a laser energy of 15 mJ on a Si-wafer in simulated martian atmospheric conditions in the UV-range (A) and in the Vis-NIR-range (B). The most intense emission lines in the UV-range are Si(III) at 254.2 nm, several Si(I) lines between 250-253 nm, and Si(I) at 288 nm followed by carbon emission from the atmosphere. For 15 mJ, C(III) at 229.7 nm (C) is of similar intensity than the C(I) emission at 247.8 nm (D). In the Vis-NIR-range (B) the most intense emission lines are Si(II) at 385.6 nm, 413.4 nm, 505.6 nm, and 634.7 nm. There is no intense C emission in this spectral range, the most intense being two C(II) lines at 658 nm close to the H(I) emission (E). Oxygen emission from the atmosphere can be observed at the O(I) triplet at 777 nm and at O(I) 844 nm. In (C)-(E), close-ups of the carbon emission in the UV and Vis-NIR are shown for different laser energies.

Figure 2: The total emission of the and the Vis-NIR-range (272-850 nm) is shown when varying the laser energy on the sample's surface (Si-wafer). Error bars correspond to the sample standard deviation obtained from 5 measurements each. The total emission increases linearly in both spectral ranges for small energies but only continues increasing linearly for high energies in the UVrange. Figure 5, top: Ratio of the neutral Si emission lines in the UV-range from the Si-wafer to the neutral C emission at 247.8 nm from the atmosphere. This ratio varies up to a factor of 2 over the investigated energy range and is most sensitive for energies below 20 mJ. The error bars are the sample standard deviation of the five measurements per laser energy. Bottom: Ratio of neutral Si and H to neutral O at 777.2 nm from the Vis-NIR-range. Again, most variation is found for the lower laser energies and a minimum is obtained close to 30 mJ. The data obtained with 22 mJ for the Vis-NIR-range might be an outlier, cf. Fig. 4. The H to O ratio follows a different trend with a maximum between 20-30 mJ and varies more than a factor of two.

Figure 6: Total emission of the UV-range (191-374 nm) and the Vis-NIR-range (272-850 nm) recorded at different times after plasma initiation (delay time) from the Si-wafer. Error bars correspond to the sample standard deviation obtained from 5 measurements each. In the very early stages of the plasma the total emission is higher in the Vis-NIR range but decaying to a lower value than the total emission of the UVrange for delay times longer than about 300 ns. Figure 15: Total LIBS plasma emission intensity for both spectral ranges obtained on different substrate materials with error bars corresponding to the sample standard deviation obtained from 5 measurements. The total intensity is mostly higher in the UV-range except for the aluminum target.

Figure 16: LIBS spectra obtained from a pure Fe sputter target in Earth atmosphere (dark blue), Helium (gray), and martian analogue gas mixture (orange) all at a pressure of 6.5 mbar. Fe(II) emission lines relative intensity indices as listed in the NIST database are marked with an asterisk and are assigned to the axis on the right side. For the relative intensity indices, the wavelength values for transmission through air were used and shifted by -0.01 nm to match the emission line positions in the measured spectra. In the very early stages of the plasma the total emission is higher in the Vis-NIR range but decaying to a lower value than the total emission of the UVrange for delay times longer than about 300 ns. Figure 16: LIBS spectra obtained from a pure Fe sputter target in Earth atmosphere (dark blue), Helium (gray), and martian analogue gas mixture (orange) all at a pressure of 6.5 mbar. Fe(II) emission lines relative intensity indices as listed in the NIST database are marked with an asterisk and are assigned to the axis on the right side. For the relative intensity indices, the wavelength values for transmission through air were used and shifted by -0.01 nm to match the emission line positions in the measured spectra.

Figure 3 :

 3 Figure 3: The emission line intensities of carbon from the atmosphere in the UV-range are shown for different laser energies (top). Error bars correspond to the sample standard deviation obtained from 5 measurements each. The emission lines show different growth behavior with energy due to their different ionization states. Similar trends are observed for the Si emission lines from the Si wafer (bottom).

Figure 4 :

 4 Figure 4: Prominent emission lines in the Vis-NIR-range of the elements of the CO 2 atmosphere and hydrogen are shown as a function of the laser energy (top).Error bars correspond to the sample standard deviation obtained from 5 measurements each. The data obtained with 22 mJ is not following a smooth trend and might be an outlier due to suboptimal measurement performance. Silicon emission lines of different ionization states in the Vis-NIR-range from the Si-wafer show a similar growth behavior (bottom).

Figure 7 :

 7 Figure 7: The emission line intensities of carbon from the atmosphere in the UV-range are shown for different delay times after plasma initiation (top).Error bars correspond to the sample standard deviation obtained from 5 measurements each. Emission of higher ionized particles decreases faster while emission from neutral particles continues to the later stages in the cooling plasma. Similar trends are observed for the Si emission lines from the Si wafer (bottom). The trends are similar for emission lines from particles with the same ionization degree.

Figure 8 :

 8 Figure8: Emission line intensities in the Vis-NIR-range of carbon and oxygen from the atmosphere and hydrogen at different delay times after plasma initiation (top). Error bars correspond to the sample standard deviation obtained from 5 measurements each. As seen in the UV-range, the emission of ionized particles decays shortly after plasma formation. Similar trends are observed for the silicon emission lines from the silicon-wafer (bottom).

Figure 9 :

 9 Figure9: Neutral silicon emission line intensity from the Si-wafer normalized by neutral atmospheric carbon from the UV-range (top) and neutral silicon and hydrogen normalized with the emission line intensity of neutral oxygen in the Vis-NIR-range (bottom). Different trends and variations can be seen, in particular a dip for early plasma life times in the ratio of silicon to oxygen in the NIR, indicating a timing discrepancy of these emissions.

Figure 10 :

 10 Figure10: Total LIBS plasma emission intensity for both spectral ranges on pressed silicon grains with different grain sizes. Error bars correspond to the sample standard deviation obtained from 5 measurements each. Highest intensity is obtained from the targets with smallest grain sizes < 25µm.

Figure 11 :

 11 Figure 11: Ratio of neutral Si to C in the UV-range (top) and neutral Si and H to O in the Vis-NIR-range (bottom) on pressed silicon grains with different grain sizes. There is high variation in the data obtained from different positions on the same sample and no strong trends were observed with the grain size.

Figure 12 :

 12 Figure 12: Emission line intensities of neutral silicon and carbon in the UV range from 5 different positions on the quartz sample are shown (top). The ratio of neutral silicon to neutral carbon is shown below.

Figure 13 :

 13 Figure13: Neutral carbon emission from a mars-analogue atmosphere at 247.9 nm obtained on different pure metals and metalloids. Many Fe(II) emission lines are present in this wavelength region as well.

Figure 14 :

 14 Figure 14: Emission line intensities of ionic and neutral carbon and neutral oxygen obtained on the different pure metal and metalloid substrate materials. Error bars correspond to the sample standard deviation obtained from 5 measurements each.

Figure 1 :

 1 Figure 1: High-resolution LIBS spectra of a Si-wafer obtained with a laser energy of 15 mJ on a Si-wafer in simulated martian atmospheric conditions in the UV-range (A) and in the Vis-NIR-range (B). The most intense emission lines in the UV-range are Si(III) at 254.2 nm, several Si(I) lines between 250-253 nm, and Si(I) at 288 nm followed by carbon emission from the atmosphere. For 15 mJ, C(III) at 229.7 nm (C) is of similar intensity than the C(I) emission at 247.8 nm (D). In the Vis-NIR-range (B) the most intense emission lines are Si(II) at 385.6 nm, 413.4 nm, 505.6 nm, and 634.7 nm. There is no intense C emission in this spectral range, the most intense being two C(II) lines at 658 nm close to the H(I) emission (E). Oxygen emission from the atmosphere can be observed at the O(I) triplet at 777 nm and at O(I) 844 nm. In (C)-(E), close-ups of the carbon emission in the UV and Vis-NIR are shown for different laser energies.

Figure 2 :

 2 Figure2: The total emission of the and the Vis-NIR-range (272-850 nm) is shown when varying the laser energy on the sample's surface (Si-wafer). Error bars correspond to the sample standard deviation obtained from 5 measurements each. The total emission increases linearly in both spectral ranges for small energies but only continues increasing linearly for high energies in the UVrange.

Figure 3 :

 3 Figure 3: The emission line intensities of carbon from the atmosphere in the UV-range are shown for different laser energies (top). Error bars correspond to the sample standard deviation obtained from 5 measurements each. The emission lines show different growth behavior with energy due to their different ionization states. Similar trends are observed for the Si emission lines from the Si wafer (bottom).

Figure 4 :

 4 Figure 4: Prominent emission lines in the Vis-NIR-range of the elements of the CO 2 atmosphere and hydrogen are shown as a function of the laser energy (top). Error bars correspond to the sample standard deviation obtained from 5 measurements each. The data obtained with 22 mJ is not following a smooth trend and might be an outlier due to suboptimal measurement performance. Silicon emission lines of different ionization states in the Vis-NIR-range from the Si-wafer show a similar growth behavior (bottom).

Figure 5 ,

 5 Figure5, top: Ratio of the neutral Si emission lines in the UV-range from the Si-wafer to the neutral C emission at 247.8 nm from the atmosphere. This ratio varies up to a factor of 2 over the investigated energy range and is most sensitive for energies below 20 mJ. The error bars are the sample standard deviation of the five measurements per laser energy. Bottom: Ratio of neutral Si and H to neutral O at 777.2 nm from the Vis-NIR-range. Again, most variation is found for the lower laser energies and a minimum is obtained close to 30 mJ. The data obtained with 22 mJ for the Vis-NIR-range might be an outlier, cf. Fig.4. The H to O ratio follows a different trend with a maximum between 20-30 mJ and varies more than a factor of two.

Figure 6 :

 6 Figure 6: Total emission of the and the Vis-NIR-range (272-850 nm) recorded at different times after plasma initiation (delay time) from the Si-wafer. Error bars correspond to the sample standard deviation obtained from 5 measurements each. In the very early stages of the plasma the total emission is higher in the Vis-NIR range but decaying to a lower value than the total emission of the UVrange for delay times longer than about 300 ns.

Figure 7 :

 7 Figure 7: The emission line intensities of carbon from the atmosphere in the UV-range are shown for different delay times after plasma initiation (top). Error bars correspond to the sample standard deviation obtained from 5 measurements each. Emission of higher ionized particles decreases faster while emission from neutral particles continues to the later stages in the cooling plasma. Similar trends are observed for the Si emission lines from the Si wafer (bottom). The trends are similar for emission lines from particles with the same ionization degree.

Figure 8 :

 8 Figure 8: Emission line intensities in the Vis-NIR-range of carbon and oxygen from the atmosphere and hydrogen at different delay times after plasma initiation (top). Error bars correspond to the sample standard deviation obtained from 5 measurements each. As seen in the UV-range, the emission of ionized particles decays shortly after plasma formation. Similar trends are observed for the silicon emission lines from the silicon-wafer (bottom).

Figure 9 :

 9 Figure 9: Neutral silicon emission line intensity from the Si-wafer normalized by neutral atmospheric carbon from the UV-range (top) and neutral silicon and hydrogen normalized with the emission line intensity of neutral oxygen in the Vis-NIR-range (bottom). Different trends and variations can be seen, in particular a dip for early plasma life times in the ratio of silicon to oxygen in the NIR, indicating a timing discrepancy of these emissions.

Figure 10 :

 10 Figure 10: Total LIBS plasma emission intensity for both spectral ranges on pressed silicon grains with different grain sizes. Error bars correspond to the sample standard deviation obtained from 5 measurements each. Highest intensity is obtained from the targets with smallest grain sizes < 25µm.

Figure 11 :

 11 Figure 11: Ratio of neutral Si to C in the UV-range (top) and neutral Si and H to O in the Vis-NIR-range (bottom) on pressed silicon grains with different grain sizes. There is high variation in the data obtained from different positions on the same sample and no strong trends were observed with the grain size.

Figure 12 :

 12 Figure 12: Emission line intensities of neutral silicon and carbon in the UV range from 5 different positions on the quartz sample are shown (top). The ratio of neutral silicon to neutral carbon is shown below.

Figure 13 :

 13 Figure 13: Neutral carbon emission from a mars-analogue atmosphere at 247.9 nm obtained on different pure metals and metalloids. Many Fe(II) emission lines are present in this wavelength region as well.

Figure 14 :

 14 Figure 14: Emission line intensities of ionic and neutral carbon and neutral oxygen obtained on the different pure metal and metalloid substrate materials. Error bars correspond to the sample standard deviation obtained from 5 measurements each.

Figure 15 :

 15 Figure15: Total LIBS plasma emission intensity for both spectral ranges obtained on different substrate materials with error bars corresponding to the sample standard deviation obtained from 5 measurements. The total intensity is mostly higher in the UV-range except for the aluminum target.

Acknowledgement

We gratefully thank P.-Y. Meslin (IRAP, Toulouse, France) for fruitful discussions.

The relative intensities cannot be compared directly between different elements and species (degree of 834 ionization). 835


Jean-Philippe Bastard

Soraya Fellahi

Axel Regeniter

Jacqueline Capeau

Pierre Ronco

Emmanuelle Plaisier

Aside from acute renal failure cases, are urinary markers of glomerular and tubular function useful in clinical practice?

Keywords: proteinuria, glomerular disease, tubulopathy

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Proteinuria is a major biomarker of kidney diseases. In physiological conditions, the glomerular filtration barrier is impermeable to plasma proteins with a molecular weight over 60 kD, including albumin, transferrin and immunoglobulin G, whereas low molecular weight proteins (alpha-1 microglobulin, retinol binding protein, free light chains…) freely reach the primary urine formed after the glomerular ultrafiltration and are reabsorbed by the proximal tubule, leading to a final urine protein-creatinine ratio under 20 mg/mmol. The qualitative evaluation of proteinuria represents a crucial diagnostic step in clinical practice for the classification of renal diseases according to glomerular, tubulo-interstitial, mixed injury or related to monoclonal gammopathy. Combined with the quantitative evaluation, it also allows an assessment of the disease's severity and prognosis as well as the response to treatment (1 -4). Several tools are currently available to evaluate proteinuria (Table 1). Urine dipstick allows a semi quantitative assessment of glomerular proteinuria by the specific detection of albuminuria but it is not informative for tubulo-interstitial proteinuria or for abnormal excretion of monoclonal free light chains (FLC). Total urine protein measurement addresses the quantitative evaluation and albuminuria excretion is informative for the presence of prominent glomerular lesions when urine albumin/protein ratio exceeds 0.6. Specific assays for low molecular weight urine makers are also available in clinical use, including alpha-1-microglobulin, retinol binding protein or bêta-2-microglobulin. Except the detection of urine monoclonal FLC, the usefulness of urine protein electrophoresis (UPEP) remains limited because it only provides semiquantitative and semi-qualitative information. Several urine biomarker assays have been developed in recent years that specifically address acute kidney injury (AKI) diagnosis and prognosis and for which abundant reports are available (5 -8). These biomarkers mostly include tubular proteins such as urinary neutrophil gelatinase associated lipocalin, urinary kidney injury molecule-1, urinary livertype fatty acid binding protein, urinary interleukin-18, urinary cystatin C, and the product of urinary tissue inhibitor of metalloproteinase-2 and insulin-like growth factor binding protein 7 (5 -8).

Although their pathophysiological relevance in AKI has been demonstrated, their clinical use remains to be established.

The development of the urine protein profile (UPP) combines specific urine protein assays on a urine spot, analyzing glomerular protein markers such as albumin, transferrin and immunoglobulin G (IgG), and tubular markers such as alpha-1-microglobulin and retinol binding protein, to generate a detailed quantitative and qualitative proteinuria assessment. While FLC are not specifically assessed in UPP, abnormal urine FLC excretion may be suspected, and should be confirmed by urine electrophoresis and/or urine immunofixation (4). The software "MDI Lablink", developed by Regeniter et al., allows a classification of proteinuria integrating these marker proteins (1 -4). In this mini-review we illustrate the advantages of using the UPP and the software.

(1) Technical considerations and interpretations UPP is performed on a minimal volume (0.5 ml) of unconcentrated urine sample, ideally resulting from the second morning urine. The proteins are measured by immunonephelometry, a specific and analytically very sensitive method that can measure proteins reliably down to 0.3 mg/l. The result is usually available on the day of collection. Urine marker evaluation by UPP is a two-step process: [START_REF] Regeniter | Interpreting complex urinary patterns with MDI LABLINK: a statistical evaluation[END_REF] Step 1 : measurement of total protein, urine creatinine, and two protein markers testing either glomerular (albumin) or tubular (alpha-1-microglobulin) dysfunction, respectively, and the urinary dipstick which estimates erythrocyte, white blood cell and nitrite count [START_REF] Maachi | Patterns of proteinuria: urinary sodium dodecyl sulfate electrophoresis versus immunonephelometric protein marker measurement followed by interpretation with the knowledge-based system MDI-LabLink[END_REF] Step 2 : only performed if albumin to creatinine ratio (ACR) and/or alpha-1-microglobulin to creatinine ratio (A1MCR) are higher than the upper normal limit, respectively > 3 mg/mmol and 2.26 mg/mmol (9 -10): additional urine protein makers are evaluated to classify abnormal proteinuria. Transferrin confirms the glomerular alteration and IgG is indicative of severe damage of the glomerular filtration barrier.

Tubulo-interstitial alterations, suspected by an increase of A1MCR, are further evaluated by retinol binding protein, which is particularly elevated in cases of severe proximal tubular cell dysfunction and when tubulo-interstitial fibrosis is present. UPP results are presented graphically (Fig. 1-Fig. 5) with a diagnosis commentary generated by the MDI Lablink software, and an additional graph that show historical results when available.

(2) UPP in the clinical practice UPP is indicated if a total proteinuria/creatinine ratio over 30 mg/mmol is detected, and/or for the evaluation of patients with a glomerular filtration rate (GFR) decrease below 60 ml/min/1.73m 2 or if the patients are exposed to a specific renal risk, including nephrotoxic or extrarenal conditions such as infection, systemic diseases.

We propose to illustrate the diagnostic and prognostic usefulness of UPP in different common clinical situations

UPP in glomerular diseases

While glomerular diseases are typically characterized by proteinuria composed by more than 60% of albuminuria, additional evaluation of urine IgG and protein tubular makers may be informative for the non-invasive evaluation of glomerular alterations and associated tubulo-interstitial lesions.

Cases 1, 2 and 3 illustrate how UPP anticipate distinct histopathological glomerular defects and associated tubulo-interstitial lesions in patients presenting with oedema and nephrotic syndrome. In addition, UPP provides an important diagnostic and prognosis orientation in patients with nephrotic syndrome and eGFR decrease, to distinguish functional pre renal azotemia to progression of glomerular and tubular lesions.

Case 1 (Fig. 1): A 21-year old woman presented with an idiopathic nephrotic syndrome related to histologically-characterized minimal change disease (MCD) with normal GFR. Protein-creatinine ratio (PCR) at diagnosis was 446 mg/mmol, with ACR and transferrin creatinine ratio at 310 and 27 mg/mmol respectively, while IgG was normal, indicative of glomerular proteinuria of high selectivity.

Alpha-1-microglobulin and retinol binding protein were around or below the normal limit. Two weeks after steroids administration, a complete remission was observed with both PCR, ACR below the normal limit at 1 and < 1.6 mg/mmol. In most of the MCD relapse, urine protein profile show selective proteinuria. However, the use of UPP anticipates distinct histopathological glomerular defects and associated tubulo-interstitial lesions in patients presenting with oedema and nephrotic syndrome in the follow-up. Thus, such a UPP (Fig. 1) during MCD relapse, similar to the UPP at diagnosis may allow avoiding the realization of kidney biopsy since it permits distinguishing a "simple" MCD relapse compared to a progression of glomerular and/or tubular lesions in this context.

In most of the MCD relapse, urine protein profile show selective proteinuria. However, the use of UPP anticipates distinct histopathological glomerular defects and associated tubulo-interstitial lesions in patients presenting with oedema and nephrotic syndrome in the follow-up. In this case, the same UPP during MCD relapse may avoid the need for kidney biopsy by allowing distinguishing between a "simple" MCD relapse and a progression of glomerular and/or and additional tubular lesions before a symptomatology of nephrotic syndrome Case 2 (Fig. 2): A 23-year old man presented with acute edema and acute kidney injury with GFR decrease. A nephrotic syndrome was detected with protein-creatinine ratio (PCR) of 849 mg/mmol, and non-selective glomerular proteinuria with ACR of 542 mg/mmol, transferrin -creatinine ratio of 57 mg/mmol and an increase of IgG-creatinine ratio of 54 mg/mmol. In addition, alpha-1microglobulin and retinol binding protein were dramatically increased (Fig. 2). Renal biopsy showed focal and segmental glomerulosclerosis responsible for the massive non selective glomerular proteinuria, but also severe lesions of acute tubular injury that were suspected by the presence of increased urine tubular markers. Case 3: A 64-year old man was referred for nephrotic syndrome with preserved GFR of 70 ml/min/1.73m 2 . UPP at diagnosis showing a non-selective glomerular proteinuria and a renal biopsy established the diagnosis of primary membranous glomerulopathy without tubular alteration or interstitial fibrosis. At 12 months, whereas the PCR was unchanged, UPP profile was modified with an increased urine IgG suggesting a progression of the glomerular disease (GFR= 25 ml/min/1.73m 2 ), as well as a huge increase of both tubular makers alpha-1-microglobulin and retinol binding protein (4).

A second kidney biopsy indeed demonstrated a progression of the glomerular lesions and the development of chronic tubulointerstitial changes related to the uncontrolled glomerular disease, as suspected by UPP. This highlights the importance of specific tubular and glomerular protein assays since the monitoring of such nephropathy on only protein and albumin assays is not sufficiently informative.

UPP in renal tubulo-interstitial diseases

Non-invasive detection of tubular and/or interstitial renal lesions may be very challenging. Indeed, urine dipstick only detects albumin and UPEP provides little information on low molecular weight proteins. It is important to notice that tubulopathies generally present with low grade proteinuria with PCR below 0.1 g/mmol. The reference range of tubular proteins is very low (retinol binding protein < 0.75 mg/g creatinine (0.08 mmol/l), alpha-1-microglobulin <14 mg/g creatinine (1.58 mmol/l)) and the chemical measurement method of total protein does not detect these proteins adequately. Tubulopathies can be observed with PCR at 0.2 to 0.3 g/mmol. In addition, follow-up evaluation after specific therapeutic intervention must be adequately assessed by specific longitudinal quantitative evaluation of tubular markers.

Case 4 (Fig. 3): This is the case of a patient hospitalized for severe impairment of GFR in the context of systemic sarcoidosis. Total proteinuria was below the limit of detection of the assay method.

However the use of UPP in this context showed an increase of the alpha-1-microglobulin with a normal ACR. The realization of the complete UPP thus showed tubulointerstitial proteinuria with normal proteinuria and albuminuria (FIG. 3). Renal biopsy showed severe granulomatous interstitial nephritis. This example focuses on the extended ability of UPP to detect early renal abnormalities when tubular marker proteins are measured. The only alternative for detecting these disorders is invasive renal biopsy. Indeed, acute estimated GFR decrease in sarcoidosis may be related to acute kidney injury induced by acute interstitial nephritis (AIN), or to pre-renal azotemia related to hypercalcemia or both. Diagnostic and therapeutic interventions are quite distinct with kidney biopsy only required in suspected interstitial inflammation. Tis case illustrates that detection of abnormal increase of retinol binding protein in addition to alpha-1-microglobulin strongly suggests AIN, a diagnosis that may be mistaken by urine PCR within the normal range.

Case 5: A HIV-infected patient, receiving Tenofovir, was referred for severe acute kidney injury with GFR of 10 ml/min/1.73m 2 and PCR= 86 mg/mmol. UPP showed 15 and 22 x fold increase of alpha-1microglobulin and retinol binding protein, respectively, while ACR was 11 mg/mmol. Tubular proximal toxicity of Tenofovir was suspected and confirmed after the rapid decrease and complete normalization of the both tubular marker, 2 months after drug discontinuation.

UPP in renal complications of monoclonal gammopathy

A wide range of glomerular and tubulo-interstitial alterations may be observed during monoclonal gammopathy, mostly related to the nephrotoxicity of the monoclonal FLC or heavy chain. Myeloma cast nephropathy is the most frequent renal complication in multiple myeloma, related to intratubular precipitation of monoclonal FLC present in large excess in urines, and early diagnosis and therapeutic intervention are required to avoid non-reversible end stage renal disease. When the sum of specific marker proteins is very low compared to the total protein, the sample should be checked for free urinary light chains (Bence Jones protein). Case 6: A 73-year old woman was referred for acute kidney injury (GFR=28 ml/min) associated with severe anemia. UPP the day of admission showed PCR at 492 mg/mmol with ACR of 11 mg/mmol, an increase of tubular makers with A1MCR = 14.6 mmol/l and RBPCR = 1.09 mg/mmol (Fig. 4); urine excess of monoclonal FLC was suspected regarding the discrepancy between PCR and albuminuria, confirmed by urine immunofixation and the diagnosis of multiple myeloma was established.

Chemotherapy combining dexamethasone, cyclophosphamide and bortezomib was promptly initiated two days after admission, leading to a dramatic decrease of PCR, tubular marker normalization within 4 weeks (Fig. 5) and huge GFR improvement.

In conclusion UPP presents several advantages. First of all, one of the major advantages is that it integrates tubular biomarkers in its analysis, which are tools that were lacking until their development in clinical practice. Second, the use of several different urinary biomarkers is useful in glomerular disease to evaluate severity of glomerular barrier damage at diagnosis and follow-up; Third, UPP allows us a precise quantification of tubular proteinuria as compared to UPE that only allows a semi quantitative evaluation. Moreover, UPP with complete evaluation available in 2 hours if necessary is less time consuming than UPE in addition to bêta-2-microglobulin, total proteinuria and albuminuria measurements, which is a major consideration in cases of acute kidney injury that usually required rapid etiologic orientation. In addition, the determination of specific urinary proteins measured on a nephelometer makes it easy to integrate this activity into that of a conventional biochemistry laboratory. This makes it possible to avoid the systematic use of electrophoresis of urinary proteins, which requires an additional technical station dedicated to its implementation, for a simple typing of proteinuria outside the suspicion of monoclonal pathology. Thus, UPP represents a non-invasive and suitable diagnostic tool for the diagnostic and prognosis approach of the renal diseases. Results expressed as mg/mmol of creatinine as a graph allowing to appreciate urinary proteins increase compared to usual values.

Figure 1 :

 1 Figure 1: 21-year old woman presented with a relapse of idiopathic nephrotic syndrome related to

Figure 2 :

 2 Figure 2: 23-year old man with nephrotic syndrome presented by acute edema and acute

Figure 3 :

 3 Figure 3: Patient hospitalized for severe impairment of GFR in the context of systemic sarcoidosis.

Figure 4 :

 4 Figure 4: 73-year old woman with multiple myeloma referred for acute kidney injury (GFR=28

Figure 5 :

 5 Figure 5: 73-year old woman with multiple myeloma after treatment combining dexamethasone,

Table I :

 I Proteinuria evaluation tools in clinical practice

			Quantitative		Qualitative
				Glomerular	Tubulointerstitial Monoclonal light
						chain
	Dipstick		+/-	+/-
	Total Proteinuria	++
	Albuminuria	++	++
	Urinary protein	+/-	+/-	+/-	++
	electrophoresis
	Urine	protein	++	++	++	+/-
	profile

Acknowledgment

The authors thank Chloé McAvoy for the proofreading of this article