Jérémie Pourchez
email: pourchez@emse.fr

Sandrine Parisse

Gwendoline Sarry

Sophie Perinel-Ragey

Jean-Michel Vergnon

Anthony Clotagatide

Nathalie Prévot

Nathalie Prévôt

Impact of power level and refill liquid composition on the aerosol output and particle size distribution generated by a new-generation e-cigarette device

Keywords: Electronic cigarettes, smoking cessation, ENDS, aerosol, MMAD, cascade impactor

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

Impact of power level and refill liquid composition on the aerosol output and particle size distribution generated by a new-generation e-cigarette device

INTRODUCTION

Electronic cigarettes or electronic nicotine delivery systems (ENDS) have recently exploded onto the smoking cessation scene as a newer way to deliver nicotine. Although the primary marketing focus is the use of ENDS as an alternative nicotine source, ENDS are used by some as a new nicotine replacement therapy (NRT) [START_REF] Breland | Science and Electronic Cigarettes: Current Data, Future Needs[END_REF]. By delivering aerosol nicotine, the ENDS devices have the potential to serve as a substitute for smoking cigarettes and thus as a harm reduction tool. While vaping may not be harmless, ENDS have demonstrated a much more favorable toxicological profile than cigarettes since most of the chemicals causing smoking-related disease are absent (Public health England 2015). Indeed, contrary to smoking cigarettes, ENDS do not contain tobacco and deliver nicotine-containing aerosol without any combustion.

ENDS, in all their shapes and technical designs, have already been present on the market for a decade. Basically, ENDS are battery-powered personal vaporizers. The main components are a mouthpiece, a tank for the refill liquid, a heating element, a battery and sometimes a microprocessor. The physical principle common to all ENDS is a small heating element that vaporizes a refill liquid to generate an aerosol (called "vape"). This refill liquid (called "eliquid") contains nicotine, humectants (such as glycerol and propylene glycol) and in the vast majority of cases, other ingredients in small quantities (water, ethanol, flavorings, etc.) [START_REF] Breland | Science and Electronic Cigarettes: Current Data, Future Needs[END_REF]. Since its emergence in the 2000s and throughout its relatively short history, the ENDS industry has continuously evolved. New technologies were quickly developed, thanks to various innovations and technological breakthroughs. Thus, many products have constantly been brought to market. These rapid developments in manufacturing induce a great variability of ENDS in terms of power source voltage, heating element resistance, and other technical features.

ENDS typically fall into at least three categories, from small cigarette-like devices (firstgeneration) to new-generation devices including high-capacity batteries with electronic circuits that provide high energy to a refillable atomizer (second-and third-generation). The third-generation of ENDS (also called advanced personal vaporizers) has only recently appeared. These devices encompass a wide variety of product types and brands. They have stronger batteries, variable setting features and tank-type atomizers. The tank-type atomizers consist of a chamber that contains the refill liquid and a wick-coil head. These devices generally incorporate control hardware that lets the user modify the voltage and/or wattage of the battery.

A new trend emerging in the ENDS users (called "vapers") community consists of using variable voltage/wattage ENDS associated with regulated mods (i.e., modified parts in the frame of the concept of rebuildable atomizers) to apply high energy. Recently, a study underlined specific features when "vaping" at high voltage [START_REF] Farsalinos | Protocol proposal for, and evaluation of, consistency in nicotine delivery from the liquid to the aerosol of electronic cigarettes atomizers: regulatory implications[END_REF]. Highpower ENDS can deliver high levels of aerosol nicotine [START_REF] Farsalinos | Protocol proposal for, and evaluation of, consistency in nicotine delivery from the liquid to the aerosol of electronic cigarettes atomizers: regulatory implications[END_REF]. This newer technology of ENDS can generate at high voltage aerosol nicotine in a much wider range of concentrations (2.72-10.61 mg of nicotine/20 puffs) than older ENDS devices using cartomizers (1.01-3.01 mg of nicotine/20 puffs) but also tobacco cigarettes (1.76-2.20 mg of nicotine/cigarette). Thus, high-power ENDS could be a promising technology to raise nicotine absorption and plasma nicotine levels. To relate exposure to biological effects (such as nicotine absorption), it is crucial to assess the deposition pattern of inhaled particles [START_REF] Pichelstorfer | Simulation of aerosol dynamics and deposition of combustible and electronic cigarette aerosols in the human respiratory tract[END_REF]. Many parameters are needed (e.g., aerodynamic diameter, composition of the particles, composition of the vapor phase, parameters of the mixture such as vapor pressure, surface tension, activity coefficient, etc.) to obtain a proper description of the deposition of the dynamic aerosols in the respiratory tract. In particular, the accurate determination of the particle size distribution is essential to predict aerosol deposition [START_REF] Sosnowski | Predicted Deposition of E-Cigarette Aerosol in the Human Lungs[END_REF]. However, aerodynamic features of the aerosol generated by high-power ENDS remain poorly known. Against this background, this work describes the impact of power level and refill liquid composition on aerosol features generated by recent high-power ENDS. Aerosol features were assessed in terms of Mass Median Aerodynamic Diameter (MMAD), Count Median Aerodynamic Diameter (CMAD), and aerosol output (i.e., the mass of airborne refill liquid per volume of aerosol generated by the ENDS). The purpose of this work is to remedy the lack of studies addressing:

(i)

The impact of the aerosol dilution ratio (from 0 to 6) prior to particle size distribution measurement.

(ii)

The effects of two propylene glycol (PG)/vegetable glycerin (VG) ratios (80% + 20% vs. 20% + 80%) on particle size distribution and aerosol output.

(iii)

The influence of the power level of the battery (from 7 W to 22 W) on particle size distribution and aerosol output.

METHODS

Materials and experimental design

A recent technology of high-power ENDS was used (purchased in March 2016 from a local store and online distributor). This ENDS device was made up of a variable lithium-ion battery (iStick 30W, Eleaf) and an atomizer (GS Air, Eleaf). Under the support of a 2200 mAh battery capacity, the battery produces performance between 2 V-8 V (or 5 W-30 W). The atomizer emphasizes a dual-coil atomizer head. The main technical features of the atomizer are a refill liquid capacity of 2.5 mL, a voltage in the 3.5 V-5.5 V range, a wattage in the 8 W-20 W range and a resistance of 1.5 ohm. A thread-adjustable air inflow allows the changing of the airflow resistance by adjusting the tightness of the screw. Prior to performing experiments, batteries were fully charged. In addition, the maximum air inflow position was fixed, and the electrical resistance of the atomizer was checked at 1.5 ± 1 ohm. Atomizers were regularly changed to avoid bias and to avoid using a degraded and/or dirty coil. All combinations of vaping parameters allow the limitation of the overheating of the refill liquid (i.e., the so-called "dry puff phenomenon"). Because it results in a strong unpleasant taste, the dry puff phenomenon can easily be detected by vapers and avoided in real life practice by reducing the power levels and the puff duration (Farsalinos et al. 2015b).

Two different compositions of refill liquid were used corresponding to 80% propylene glycol (PG) + 20% vegetable glycerin (VG), noted as 80PG/20VG, and 20% PG + 80% VG, noted as 20PG/80VG. These formulations were prepared in the laboratory from commercial solutions available on the market to do the refill liquid oneself (100-VG and 100-PG base, A&L, France). The refill liquids used for this study were nicotine-free and flavor-free to exhibit only the impact of the PG/VG ratio on the aerodynamic features. The rationale in selecting the compositions of the refill liquid (80/20 and 20/80) and the power level of the ENDS battery (from 7 to 22 W) is to cover a wide range including the most popular parameter combinations used by vapers. Finally, although the nominal capacity of the tank-type atomizer is 2.5 mL, the atomizer was filled with 2 mL of the prepared solution to avoid potential overfilling. For all measurements, the ambient conditions were as follows:

temperature of 23 ± 2 °C, relative humidity from 45% to 75%, pressure from 86.0 kPa to 106.0 kPa.

Particle size distribution (mass distribution and MMAD): experimental conditions using a high flow rate and measuring aerosol aged for a few seconds

Aerosol particle sizing was defined in terms of Mass Median Aerodynamic Diameter (MMAD). Our objective was to compare two measurement strategies (mass distribution vs.

count distribution using the same cascade impactor design) to examine the impact of experimental biases due to particle evaporation (because of high dilution ratios) or particle coagulation (because of long residence times between aerosol sampling and measurement).

The DLPI setup was used (Dekati Low-Pressure Impactor; Dekati Ltd., Finland) to quantify the mass distribution and MMAD. In cascade impactors, the aerosolized particles are impacted on different stages depending on their inertia related to their aerodynamic diameter.

This device operates with an air flow of 10 L•min -1 and allows the collection of nebulized particles from 7 nm to 10 µm in 12 size fractions. The DLPI design consists of a 12-stage cascade low pressure impactor leading to the determination of gravimetric size distribution.

An in-house interface was developed to reproducibly introduce a puff that was well-controlled for duration and volume into the inlet of the impactor. Based on the work of Bertholon et al. [START_REF] Bertholon | Comparison of the aerosol produced by electronic cigarettes with conventional cigarettes and the shisha[END_REF], our interface was composed of a 3-L syringe (Hans-Rudolph, USA) acting as a reservoir during the puffing and connected to the DLPI cascade impactor and the ENDS (Figure 1). Aerosol sampling was carried out considering a 4-s puff (with a flow rate of 500 mL/s) and various dilution ratios from 0 to 6 (e.g., a dilution ratio of 1.5 corresponds to 2 L of aerosol generated by the ENDS diluted in 1 L of ambient air initially present in the 3-L syringe). At the end of the 4-s puff duration, the volume of aerosol contained in the syringe was injected into the DLPI setup using the 10 L•min -1 flow rate of the cascade impactor without any other air dilution. However, the main drawback of this protocol is having a very disparate flow rate (500 mL/s) compared to the more usual flow rate (10-50 mL/s) used by vapers in real-life practice. The main advantage of using this high flow rate is to obtain a significant mass of aerosol during a short sample residence time to be able to determine the aerosol mass distribution, thanks to the size-fractionation through the DLPI setup. We must keep in mind that the ENDS aerosol is highly dynamic and unstable, showing a half-life limited to approximately 15-30 seconds, depending on the devices tested [START_REF] Bertholon | Comparison of the aerosol produced by electronic cigarettes with conventional cigarettes and the shisha[END_REF]. Four aerosol dilution ratios (from 0 to 6) applied during the sampling step were tested to investigate their impact on changes in the particle size distribution.

According to European standard method NF EN 13544-1, the MMAD of nebulized particles was determined by tracing the cumulative curve of mass vs. size. The cumulative mass was obtained using a gravimetric method consisting of weighing each DLPI stage after the experiments using an electronic precision balance (Adventurer Pro, OHAUS, USA). The MMAD was interpolated from the particle size distribution curve by noting the particle size at which the line crosses the 50 % mark. The geometric standard deviation (i.e., GSD) should be calculated only if the aerosol was log-normally distributed. The calculation of GSD was performed by noting the particle size X at which the line crosses the 84.13 % mark and the particle size Y at which the line crosses the 15.87 % mark. Then, the GSD was calculated from the equation X/Y 0.5 .

Particle size distribution (count distribution, CMAD and resulting calculated MMAD): experimental conditions using a low flow rate and freshly generated aerosol

The second measurement strategy used the ELPI set-up (Dekati Ltd., Finland). This sizing technique allows the quantification of the count distribution of the aerosol generated by highpower ENDS using the same cascade impactor used by the DLPI setup. Thus, as in the DLPI, the ELPI allows the collection of nebulized particles from 7 nm to 10 µm into 12 size fractions and operates with an air flow of 10 L•min -1 [START_REF] Järvinen | Calibration of the new electrical low pressure impactor (ELPI+)[END_REF]. The ELPI operating principle can be divided into three major parts: particle charging in a unipolar corona charger, size classification in a DLPI cascade impactor and then electrical detection with sensitive electrometers. Finally, the induced current of the impacted particles is measured and related to the aerodynamic cut-off diameter of the impactor stage. The results can be expressed by means of number concentrations to calculate the CMAD. Aerosol sampling was carried out considering a 3-s puff (with a flow rate of 20 mL/s) without aerosol

dilution. An in-house interface was designed for puffing (Figure 2). Puffs were performed using a 60-mL syringe connected to the ENDS. This syringe was also connected to the ELPI via a metal United States Pharmacopeia (USP)-like artificial throat and a PTFE mouthpiece adaptor. The USP throat (height, 112 mm; width, 42 mm; internal diameter, 19 mm) was a 90° bent metal pipe with a uniform cross section with slight contractions at the inlet and a small diffuser at the outlet. The ELPI V4.0 software recorded current vs. time data.

Experiments were performed in triplicate. Finally, CMAD and cumulative particle number distributions vs. aerodynamic diameter were obtained. Furthermore, the MMAD was calculated from count distribution. For log-normal distribution, the known relationship holds: [START_REF] Prévôt | Nicotine delivery from the refill liquid to the aerosol via high-power e-cigarette device[END_REF].

Aerosol output measurement

The aerosol output is the total mass of aerosol generated by the ENDS. The aerosol output can be measured by collecting the aerosolized refill liquid that leaves the ENDS on filters or stages of the cascade impactor. In this study, the aerosol output was expressed as mg of airborne refill liquid per liter of aerosol generated by the ENDS. Explicitly, the aerosol output was simply calculated by summing the mass of airborne refill liquid collected on the 12 stages of the DLPI for a given volume of aerosol generated by the ENDS.

RESULTS

Impact of the aerosol dilution ratio on MMAD using the DLPI measurement strategy (i.e., a high flow rate and aerosol aged for a few seconds )

To investigate the impact of the aerosol dilution ratio during the sampling step on the particle size distribution, only the results obtained for the power level fixed at 13 W and the formulation 20PG/80VG are highlighted (Table 1 with lines in gray, Figures 3 and4). A unimodal distribution was observed for all experimental conditions resulting in GSD from 1.45 ± 0.01 to 1.60 ± 0.05 (Table 1 with lines in gray). Particle sizes measured in the stage impactor patterns ranged from 0.382 µm to 2.39 µm. A significant impact of the dilution ratio on the MMAD and the aerosol output was shown. The impactor stage cutoff values resulted in 0.96 ± 0.4 µm of MMAD for no dilution, to 0.71 ± 0.2 µm of MMAD for a dilution ratio of 6

(Table 1 with lines in gray; see also Figure 3). A linear correlation (y=-0.04x+0.98, R 2 =0.96;

Figure 3) between the MMAD and the value of the dilution ratio of the aerosol generated by the ENDS was clearly observed. This decrease in both the aerosol output and the MMAD could be due to a rise in the evaporation process when the dilution ratio increases.

Nevertheless, it is impossible to exclude the possibility that there is also a contribution by coagulation (higher dilution rate means less coagulation) in a given set of experimental conditions. In addition, without dilution, the aerosol output was notably lower compared to a dilution ratio of 1.5 (13.2. ± 1.0 mg/L vs. 18.0 ± 1.2 mg/L). All things considered, the dilution ratio of 1.5 during the sampling step appeared to be the best compromise to obtain high aerosol output with, at the same time, unchanged MMAD compared to an ENDS aerosol that was not diluted.

Impact of the power level on aerosol output using the DLPI measurement strategy (i.e., high flow rate and aerosol aged for a few seconds )

Figure 5 presents the aerosol output as a function of the power level of the ENDS battery ranging from 7 W to 22 W. Unsurprisingly, the power level of the ENDS appeared to be a very important parameter playing on the aerosol output generated by ENDS. As a matter of fact, a logarithmic law seems to be quite satisfactory to predict the rise of the aerosol output when the power of the battery increases (Figure 5). Second, the impact of the refill liquid composition was also demonstrated. For a given power level of the battery, a higher aerosol output was noticed using the formulation with higher VG content compared to the formulation with higher PG content (e.g., at 16 W, 15.0 ± 1.7 mg/L for 80PG/20VG vs. 21.5 ± 1.5 mg/L for 80VG/20PG, p = 0.0172 (Mann-Whitney test), Figure 5).

Impact of the power level on the particle size distribution using the DLPI measurement strategy (i.e., high flow rate and aerosol aged for a few seconds)

Figure 6 shows the impactor-collected data by means of frequency mass distribution (cumulative mass distribution available in Figures 7 and8). Six levels of ENDS power and two formulations were investigated. Table 1 provides the particle size summary for all experimental conditions. The findings present a gradual transition of a dominant mode distributed mainly from 613 nm at 7 W to 949 nm at 22 W (Figure 6). This behavior was observed for the two VG/PG ratios studied. However, the transition from 613 nm to 949 nm seems to occur at a lower power level for the 20PG/80VG formulation compared with the 80PG/20VG formulation (i.e., 10 W for 20PG/80VG vs. 17 W/19 W for 80PG/20VG, Figure 6). As a result, a slight increase in the MMAD was noticed when the power level rises (Table 1, see also Figure 9). A linear correlation highlighting the rise of MMAD as a function of the power level was clearly evident for the 20PG/80VG formulation (y=0.02+0.61, R 2 =0.97;

Figure 9). By contrast, two linear correlations were observed (y=0.007+0.77, R 2 =0.95, in the range of 7-17 W; y=0.05+0.01, R 2 =0.99, in the range of 17-22 W; Figure 9) for the 80PG/20VG formulation with a transition at 17 W. This power level at 17 W also corresponds to the transition of a dominant mode centered on 613 nm to 949 nm for the 80PG/20VG formulation.

Impact of the power level on the particle size distribution using the ELPI measurement strategy (i.e., flow rate similar to the flow rate that vapers use in real-life practice and freshly generated aerosol)

Considering our experimental conditions (choice of ENDS device and vaping parameters such as the power range of the battery, the resistance of the atomizer, the air flow resistance adjusted on the device using the tightness of the screw), we observed that ELPI could be used for CMAD measurement of the ENDS aerosol. Our data showed a maximum count aerosol concentration measured for each puff in the range of 1.5.10 6 to 3.10 6 particles per cc. The maximum number concentration for each stage of the ELPI is always significantly lower than the maximum count aerosol concentration above 10 5 particles per cc between stages 8 and 5

(i.e., D 50 in the 0.26 μm-1 μm] range) and lower than the maximum count aerosol concentration above 10 6 particles per cc for an aerodynamic diameter lower than 0.26 μm.

For real-life conditions used by vapers (fresh aerosol and realistic flow rate), the cumulative particle number distribution was measured for the 80PG/20VG formulation and 20PG/80VG formulation (Table 2, Figure 10). In good concordance with the observations made using mass distribution data obtained with the DLPI setup for an aerosol aged for a few seconds and a high flow rate, a gradual transition of a dominant mode distributed mainly from 480 nm to 760 nm was shown when the power level rises from 7 to 22 W. A rise in the CMAD was observed between 7 and 22 W, both for the 80PG/20VG formulation and the 20PG/80VG formulation: from 0.69 ± 0.03 µm to 1.32 ± 0.06 µm and from 0.95 ± 0.05 µm to 1.28 ± 0.07 µm, respectively. To summarize, the main point is that the PG-dominant e-liquid generates smaller particles at low wattage compared to the VG-dominant e-liquid, but they converge at 22 W.

In addition, the MMAD can also be calculated from these particle number distribution data.

Unsurprisingly, a rise in the calculated MMAD was also observed between 7 and 22 W, both for the 80PG/20VG formulation and the 20PG/80VG formulation, from 0.86 ± 0.04 µm to 1.52 ± 0.07 µm and from 1.21 ± 0.06 µm to 1.50 ± 0.08 µm, respectively. However, although the same tendency was clearly observed (i.e., a rise of MMAD when the power increases) whatever the measurement strategy used, DLPI measurements lead to lower MMAD compared to ELPI measurements when measuring freshly generated aerosol with a realistic flow rate compared to real-life conditions. For the conditions at 13 W with a dilution ratio of 1.5, the DLPI measurements showed an MMAD of 0.92 ± 0.02 µm and 0.86 ± 0.01 µm for the 80PG/20VG formulation and the 20PG/80VG formulation, respectively. For the same power level conditions, the ELPI measurement showed an MMAD of 1.26 µm ± 0.07 and 1.27 ± 0.03 µm for the 80PG/20VG formulation and the 20PG/80VG formulation, respectively.

Finally, in contrast with the mass distribution data, the count distribution data allow the detection of a population lower than 100 nm, corresponding to approximately 10 % of the particle distribution count (Figure 10). The results allow us to distinguish that this population lower than 100 nm tends to disappear at high power levels (e.g., 8.4 ± 3.5 % of the count distribution is lower than 120 nm at 7 W vs. 4.3 ± 1.1 % at 22 W for the 20PG/80VG formulation; 8.6 ± 2.4 % of the count distribution is lower than 120 nm at 7 W vs. 0.35 ± 0.15 % at 22 W for the 80PG/20VG formulation; Figure 10).

DISCUSSION

Determination of the particle size distribution for the ENDS aerosol is an experimental challenge because of the high hygroscopicity and volatile nature of the particulate matter (composed largely of propylene glycol, glycerin and water). These obstacles seriously complicate accurate characterizations of ENDS aerosol measurements. Sizing techniques requiring a high degree of aerosol dilution are expected to result in significant particle evaporation and then to induce potential bias with the alteration of the particle size distribution from that provided to the vaper. For example, particle evaporation during electrical mobility measurements can be the cause of the indication of artificially small particle sizes [START_REF] Ingebrethsen | Electronic cigarette aerosol particle size distribution measurements[END_REF]). In addition, the ENDS aerosol exhibits a dynamic behavior after puffing and during inhalation. Both the particle size distribution and the particle number concentration are expected to evolve because the aerosol is subject to condensational growth, particulate matter evaporation, coagulation and particle deposition. In this study, we compared two different measurement strategies using the following: realistic or unrealistic high flow rates to generate the ENDS aerosol, the count or mass distribution to assess aerosol sizing, and analyzing fresh aerosol or aerosol aged for a few seconds. A specific advantage of the cascade impactor is that a very high dilution of the aerosol is not required before the size measurement. As a result, potential artifacts from very important particulate matter evaporation during the aerosol sampling are quite limited compared to other aerosol sizing techniques. Nevertheless, other sizing techniques could also be applied using no or low sample dilution [START_REF] Mikheev | Real-Time Measurement of Electronic Cigarette Aerosol Size Distribution and Metals Content Analysis[END_REF][START_REF] Cabot | Aerosol measurement of e-cigarettes[END_REF][START_REF] Ingebrethsen | Electronic cigarette aerosol particle size distribution measurements[END_REF].

For example, Ingebrethsen et al. [START_REF] Ingebrethsen | Electronic cigarette aerosol particle size distribution measurements[END_REF] used not only electrical mobility but also a spectral transmission method as well that does not require a dilution.

The consistency of the results obtained and the data interpretation should be discussed. Few studies have been devoted to aerosol features generated by ENDS. In addition, results found in the literature, using various types and brands of ENDS, are often contradictory.

(i) Some papers have focused on aerosol mass by avoiding the phase transition.

Particle diameters of an average mass appeared in the 500-1000 nm range when inertial sizing techniques such as cascade impaction were used (487 nm to 631 nm for Alderman et al. [START_REF] Alderman | Particle Size Distribution of E-Cigarette Aerosols and the Relationship to Cambridge Filter Pad Collection Efficiency[END_REF], from 600 to 650 nm for Bertholon et al. [START_REF] Bertholon | Comparison of the aerosol produced by electronic cigarettes with conventional cigarettes and the shisha[END_REF], from 600 to 800 nm for Kane and Rusyniak (Kane and Rusyniak 2015) and 1.03 µm for Lerner et al. [START_REF] Lerner | Environmental health hazards of ecigarettes and their components: Oxidants and copper in e-cigarette aerosols[END_REF]). To summarize, regarding the measurement of ENDS particle size distributions, there are typically two different approaches. The first approach consists mainly of avoiding coagulation by applying a relatively high dilution rate while suffering from phase transition. A second approach consists mainly of avoiding phase transition by not diluting the aerosol while suffering from coagulation. We support the conclusion that both well-defined approaches cannot tell the whole story. In our study, we compared the results obtained with two different measurement strategies using the same design for the cascade impactor. Based on both DLPI and ELPI setup measurements, the findings obtained are consistent with cascade impactor data devoted to ENDS aerosols previously published [START_REF] Alderman | Particle Size Distribution of E-Cigarette Aerosols and the Relationship to Cambridge Filter Pad Collection Efficiency[END_REF][START_REF] Kane | Measurement of e-cigarette aerosol particle size with a low flow cascade impactor[END_REF][START_REF] Bertholon | Comparison of the aerosol produced by electronic cigarettes with conventional cigarettes and the shisha[END_REF]). Indeed, using the DLPI setup, we obtained MMAD in the 0.71-1.19 µm range with the same brand of ENDS and by varying the power level and the refill liquid composition. In addition, the results obtained using the ELPI measurement strategy showed higher MMAD compared to the DLPI measurement strategy. This difference can be explained by at least 3 factors:

-The dilution of the aerosol generated by ENDS during the sampling step (dilution ratio of 1.5 for the DLPI measurements vs. no dilution for the ELPI measurements in reallife conditions).

-The flow rate used to generate the aerosol (extremely high flow rate of 500 mL/s for the DLPI measurements vs. 20 mL/s for the ELPI measurements under real-life conditions). One assumption can be that the high flow rate used to generate the aerosol for the DLPI measurements led to more evaporation compared to the realistic flow rate. The use of an extremely high flow rate (500 mL/s) compared to a more realistic flow rate (approximately 20 mL/s) used by vapers in real-life practice remains an important drawback of the DLPI measurement strategy. Indeed, our findings showed that a high flow rate can affect the particle size distribution.

-The aging time (i.e., the sample residence duration in the syringe acting as a reservoir), which is longer for the DLPI measurements (approximately 10 s including the puff duration with the 3-L syringe) compared to the ELPI measurements (approximately 4 s including the 3-s puff duration with the 60-mL syringe). Regarding the dynamic behavior of the ENDS aerosol, the fact that the DLPI measurement strategy induces the analysis of an aerosol aged for a few seconds compared to freshly generated aerosol for the ELPI measurement strategy can partially explain our results since the evaporation process can be involved during the duration of the sampling procedure using the DLPI setup, which analyzed the aerosol that had aged for a few seconds.

This work demonstrated that the DLPI cascade impactor can be used to measure the particle size distribution under controlled conditions. However, two competing processes may operate simultaneously, making DLPI data analysis very difficult. On the one hand, the high flow rate results in aerosol dilution and is therefore most likely to lead to the partial evaporation. On the other hand, long sample residence time (above 10 s) may lead to coagulation as well.

Summarizing, the proposed method and results obtained do not allow us to conclude without ambiguity what the "true" ENDS aerosol size is. As a consequence, the DLPI procedure may be used for inter-comparison of ENDS devices (to compare ENDS brands, to test several power levels of a given ENDS technology, to define the impact of the refill liquid composition, etc.). Thus, the data related to particle size distribution produced in that way do not serve as a proper input for estimating a quantitative deposition in the human lung.

Nevertheless, our results suggest that the frequency mass distribution changes according to power levels are not that dramatic (Figure 6), so it should not result in a meaningful effect of power levels on particle deposition in the respiratory tract. We support the conclusion that the methodology proposed to quantify parameters of the aerosol generated by an ENDS device is highly welcomed. All things considered, whatever the measurement strategy used, the same tendency and the same order of magnitude were observed considering MMAD change with vaping parameters. Indeed, it is still quite unknown how the MMAD is affected by the power level of the ENDS devices or the composition of the refill liquid. In a recent study, Zhao et al. [START_REF] Zhao | Effects of design parameters and puff topography on heating coil temperature and mainstream aerosols in electronic cigarettes[END_REF]) measured both mainstream aerosols and the heating coil temperature to study the effects of the ENDS design parameters and the puff topography. These authors proved that the peak heating coil temperature and the CMD increased with longer puff duration and lower puff flow rate. Even if a better knowledge of the puff topography on the aerosol features were noticed, the impact of the power level was not examined. In our study, we demonstrated that the aerosol output rose significantly when the power level increased, following a logarithmic law. Our findings are in accordance with literature [START_REF] Talih | Effects of User Puff Topography, Device Voltage, and Liquid Nicotine Concentration on Electronic Cigarette Nicotine Yield: Measurements and Model Predictions[END_REF][START_REF] Kosmider | Carbonyl compounds in electronic cigarette vapors: effects of nicotine solvent and battery output voltage[END_REF]). In addition, these results are consistent with empirical evidence and especially from vapers who aim for the largest aerosol production, commonly referred to as "cloud chasers". Second, particle size distribution was related positively to the power level following linear correlations.

Several studies using older technologies for the atomizer have demonstrated that plasma nicotine levels are lower when using ENDS compared to smoking tobacco cigarettes [START_REF] Farsalinos | Nicotine absorption from electronic cigarette use: comparison between first and newgeneration devices[END_REF](Farsalinos et al. , 2015a;;[START_REF] Hajek | Nicotine intake from electronic cigarettes on initial use and after 4 weeks of regular use[END_REF][START_REF] Dawkins | Acute electronic cigarette use: nicotine delivery and subjective effects in regular users[END_REF][START_REF] Bullen | Effect of an electronic nicotine delivery device (e cigarette) on desire to smoke and withdrawal, user preferences and nicotine delivery: randomised cross-over trial[END_REF]). Farsalinos et al. [START_REF] Farsalinos | Protocol proposal for, and evaluation of, consistency in nicotine delivery from the liquid to the aerosol of electronic cigarettes atomizers: regulatory implications[END_REF]) recently investigated the potential of highpower ENDS in nicotine delivery from the refill liquid to the aerosol. These authors demonstrated that nicotine delivery was enhanced significantly with the new generation of atomizers, especially at high power levels. Some tank-type atomizers appeared to exceed tobacco cigarettes in nicotine delivery, due mainly to different design characteristics of the atomizer and the power delivery potential of the battery. The high levels of nicotine in the aerosol from high-power ENDS could enhance nicotine absorption. Our findings proved that even if the aerosol output of high-power ENDS increases at a high voltage (perfectly in accordance with the high nicotine delivery observed by Farsalinos et al. [START_REF] Farsalinos | Protocol proposal for, and evaluation of, consistency in nicotine delivery from the liquid to the aerosol of electronic cigarettes atomizers: regulatory implications[END_REF])), this rise in aerosol output is also accompanied by an increase in the MMAD. This change in aerodynamic behavior can significantly impact aerosol deposition within the respiratory tract and ultimately nicotine absorption and plasma nicotine levels.

At present, high-power ENDS could satisfy the nicotine craving of vapers without the need to do prolonged and/or frequent puffs. However, this point cannot be simply assessed only by determining aerosol nicotine level per puff. Indeed, due to the rise of MMAD occurring at high voltage, the efficiency of the nicotine absorption can be impacted. The rate of nebulization and droplet size are well known to be critical factors in achieving nebulizer performance [START_REF] Boe | European Respiratory Society Guidelines on the use of nebulizers[END_REF]). These factors were assessed according to standards using cascade impactors in the case of drug delivery to the lung (e.g., NF EN 13544-1 used in this study). However, specifically, the DLPI device equipment does not meet current regulatory guidelines, contrary to other inertial sizing techniques such as the Andersen Cascade Impactor (ACI) and Next Generation Impactor (NGI). We assumed that when addressing ENDS testing, combined approaches associating first the assessment of aerosol nicotine delivery (as proposed by Farsalinos et al. [START_REF] Farsalinos | Protocol proposal for, and evaluation of, consistency in nicotine delivery from the liquid to the aerosol of electronic cigarettes atomizers: regulatory implications[END_REF], and second, the measurement of aerodynamic features (based on particle size distribution and MMAD determination using cascade impactors) should be carried out for inter-comparison of ENDS devices.

CONCLUSIONS

Focusing on recent high-power ENDS, the impact of the PG/VG ratio and the power level of the battery on aerosol features was determined. This work demonstrated that:

(i)

The aerosol output significantly rose when the power level increased following a logarithmic law. The PG/VG ratio had an impact on the aerosol output. The higher the VG content in the refill liquid formulation, the higher was the mass of refill liquid found in the aerosol generated.

(ii)

The mass and count size distribution were positively related to the power level, following linear correlations between MMAD and the power level in the range of 7-22 W. A moderate impact of the PG/VG ratio was observed. Changes in the Figure 6: DLPI Impactor-collected data. Impact of the power level on the frequency mass distribution for the 80PG/20VG formulation (Figure 6A) and for the 20PG/80VG formulation (Figure 6B). Experiments were performed in triplicate with an aerosol dilution ratio of 1.5.

Five power levels ranging from 7 W to 22 W were tested, but only 3 power levels are used to illustrate trends. Figure 10: ELPI Impactor-collected data. Impact of the power level on the cumulative count distribution of the 80PG/20VG formulation (Figure 10A) and the 20PG/80VG formulation (Figure 10B). Experiments were performed in triplicate with a dilution ratio of 1.5 (fresh aerosol, realistic flow rate used compared to vapers).

  Sosnowski and Kramek-Romanowska (Sosnowski and Kramek-Romanowska 2016) found a mass median diameter of droplets emitted from ENDS at 410 nm when an optical technique such as laser diffraction was used. (ii) Some papers have focused on the number of particles by avoiding severe coagulation. Particle diameters of an average count appeared in the 30-200 nm range when electrical mobility analyzers were used (186-198 nm for Sahu et al. (Sahu et al. 2013), 120-180 nm for Zhang et al. (Zhang et al. 2013), 120-165 nm for Fuoco et al. (Fuoco et al. 2014), 107-165 nm for Manigrasso et al. (Manigrasso et al. 2015) and 18-29 nm for Zhao et al. (Zhao et al. 2016)).

Figure 1 :

 1 Figure 1: Scheme of the experimental setup for the mass sizing distribution of the aerosol

Figure 2 :

 2 Figure 2: Scheme of the experimental setup for the count sizing distribution of aerosol

Figure 3 :

 3 Figure 3: Impact of different dilution ratios of the aerosol generated by the ENDS on the

Figure 4 :

 4 Figure 4: Impact of the dilution ratios during the sampling step on the aerosol output. The

Figure 5 :

 5 Figure 5: Impact of the power level of the ENDS battery on the aerosol output. Dilution ratio

Figure 7 :

 7 Figure 7: DLPI Impactor-collected data for the 80PG/20VG formulation. Impact of the power

Figure 8 :

 8 Figure 8: DLPI Impactor-collected data for the 20PG/80VG formulation. Impact of the power

Figure 9 :

 9 Figure 9: Impact of the power level of the ENDS battery on the MMAD. Dilution ratio fixed

Table 1 :

 1 Summary of the size distribution and the aerosol output data. Experiments were performed in triplicate. MMAD refers to the Mass Median Aerodynamic Diameter. GSD refers to the Geometric Standard Deviation. The aerosol output refers to the mass of airborne refill liquid by volume of aerosol generated by the ENDS. Gray lines correspond to the impact of the dilution ratio on the main aerosol features (power level was fixed at 13 W, 20PG/80VG).

	Formulation at 20PG/80VG

Table 2 :

 2 Summary of the count distribution data. Experiments were performed in triplicate (fresh aerosol, realistic flow rate compared to vapers). CMAD refers to the Count Median Aerodynamic Diameter. GSD refers to the Geometric Standard Deviation. The MMAD was calculated from the count distribution. For log-normal distribution, the known relationship holds: MMAD = CMAD exp [3 (log (GSD)) 2 ].

	Formulation at 20PG/80VG

power level allow the transition between a dominant mode centered on 613 nm of MMAD to a mode centered on 949 nm of MMAD. These findings provide a better understanding of how high-power ENDS generates particles.

The protocol designed by [START_REF] Farsalinos | Protocol proposal for, and evaluation of, consistency in nicotine delivery from the liquid to the aerosol of electronic cigarettes atomizers: regulatory implications[END_REF] to measure nicotine delivery to the aerosol and the protocol designed in this work to measure MMAD (based on the standard applied to medical nebulizers) could be combined to serve for inter-comparison of ENDS devices.

FUNDING

There is no source of support for this study (grants, gifts, equipment or drugs) to declare.

DECLARATION OF INTERESTS

The authors are independent from the tobacco industry and declare no financial relationships with any organizations that might have an interest in the submitted work. Specifically, we do not have a source of support to declare from the company that sells the electronic cigarettes used for the submitted work.

AUTHOR CONTRIBUTIONS

Conceived and designed the experiments: JP and NP.

Performed the experiments: JP SP GS AC and NP.

Analyzed the data: JP SP GS SPR JMV AC NP.

Contributed reagents/materials/analysis tools: SP AC NP.

Wrote the paper: JP. All authors reviewed and approved the manuscript.


Marc Vérin

Pascal Benquet

The Move: when Neurosciences teach us to better teach Neurosciences

HAL is

A C C E P T E D M A N U S C R I P T

Although lecturing has been the predominant mode of instruction since the foundation of universities in Western Europe over most of a millennium ago, the traditional "teaching by telling" approach is now being undermined by the latest neuroscience and educational science data. In a well-designed recent meta-analysis published by [START_REF] Freeman | Active learning increases student performance in science, engineering, and mathematics[END_REF], 158 studies of student performance under active learning were compared with 67 studies under traditional lecturing. First, the hub-and-spoke model of semantic cognition suggests that multimodal, sensorimotor, cognitive and emotional engagement, as well as personal engagement, constitute the core 'ingredients' for constructing concepts. Active and multimodal memory processes are significantly more efficient than passive and modality-specific processes (Lambon [START_REF] Ralph | The neural and computational bases of semantic cognition[END_REF]. At neuronal and synaptic level, multiple inputs such as sensorimotor, cognitive and emotional stimuli might contribute to induce heterosynaptic plasticity by cooperativity and promote long-term potentiation and, at cognitive point of view, long-term retention.

Second, observing the symptoms to recognize and reproduce them mobilizes directly the mirror neuron system [START_REF] Rizzolatti | The mirror mechanism: a basic principle of brain function[END_REF] and promotes empathy (de Waal et al, 2017), an essential capacity for future clinicians. Moreover, the activation of the mirror neuron system is clearly superior if the action seen has already been personally experienced by the subject himself. Thus the prior imitation of a symptom favors its later both recognition and understanding of the suffering it induces [START_REF] Schütz-Bosbach | Perceptual resonance: action-induced modulation of perception[END_REF].

Third, most stages of learning are influenced by a positive emotional context (attention, detection of novelty, memorization, motivation, choice and decision). Conversely, negative emotions have a deleterious influence on learning of neutral information (such as general knowledge). Thus anxiety has a negative impact on attention, memory and metacognition (Arnstem, 2015). The friendly context of the Move program and the competition in order to win student battles mobilize together the dopaminergic reward system and promotes motivation, desire to do, attention and memorization. In the same way, the fun context and the friendly competition also favor the pleasure and the desire to do again. So liking and wanting are favored positively by this program [START_REF] Colasanti | Endogenous opioid release in the human brain reward system induced by acute amphetamine administration[END_REF]. Moreover, mobilization

A C C E P T E D M A N U S C R I P T

of the dopaminergic reward system contributes to promote both short and long term motivation [START_REF] Howe | Prolonged dopamine signalling in striatum signals proximity and value of distant rewards[END_REF][START_REF] Westbrook | Dopamine Does Double Duty in Motivating Cognitive Effort[END_REF].

The use of neuroscientific data is currently "à la mode" in the field of educational sciences. In the context of medicine learning, especially neurology, well conducted studies remain unfortunately rare. However, neuroscientists are particularly well placed to build this type of study, with a look at both innovative, because they are at the origin of the concepts newly highlighted, and critical regarding the cognitive processes and neuroanatomical substrates envisaged.

As a tribute to Jean-Martin Charcot, the inventor of Neurology, we must therefore thank Professor Emmanuel Roze's team for having taken inspiration from this great precursor both to put into practice the most modern concepts of learning, and to demonstrate their efficiency. Neurosciences still have a lot to learn from neurosciences themselves.

A C C E P T E D M A N U S C R I P T

  The results demonstrate that active learning increases both performance and motivation, and decreases failures. As Louis Cozolino points out in his book « The Social Neuroscience of Education » (2013): « While teachers may focus on what they are teaching, evolutionary history and current neuroscience suggest that it is who they are and the emotional environment in the classroom they are able to create that are the fundamental regulator of neuroplasticity ». The same conclusions are certainly applicable to the teaching of clinical neurosciences. Hundred thirty years after the important contribution of Jean-Martin Charcot in La Salpêtrière to education in neurology by changing abstract teaching into concrete teaching by putting his patients on stage during the lecture, Roze et al

have implemented in the same place a novel learner friendly program: « The Move ». To modernize the teaching approach while making it accessible and fun for students, staging elements of the TV show The Voice were incorporated (such as teams, battles, coaches, and performing in front of a jury). The Move belongs to a broad category of simulation-based learning. Comparing the performance of the students that participated in The Move program with a standard education group of students, The Move clearly improves medical student's long-term retention and delayed recall (30 months) of neurological semiology. Why is the Move program so efficient, and why is it particularly relevant for learning clinical neurosciences? Modern neurosciences who are interested in the mechanisms of learning provide strong clues to understanding the efficiency of innovative programs such as The Move.


A new non-invasive approach is proposed to study joint motions. It is based on dynamic tracking of the skin shape. A robust simultaneous registration algorithm (Iterative Median Closest Point) is used to follow the evolving shape and compute the rigid motion of the underlying bone structures. This new method relies on the differentiation of the rigid and elastic parts of the shape motion. A skin marker network is tracked by a set of infrared cameras. Unlike usual techniques, the algorithm tracks the instantaneous polyhedral shape embedding this network. This innovating approach is expected to minimize bias effect of skin sweeps and give some new information about the underlying soft tissue activities. Current application addresses the motion of the shoulder complex (humerus, clavicle and scapula). It is compared with two marker-based methods published in the literature. Preliminary results show significant differences between these three approaches. The new approach measurements give rise to greater rotations.

I. INTRODUCTION

HE upper limb has been less studied than the lower limb in the literature, because of its complexity. The functional understanding of the shoulder is still incomplete. However, a better comprehension of its motions could contribute, to obtain a better understanding of some particular trauma (e.g. shoulder impingement syndrome), to improve the conception of arm prosthesis, or to develop more efficient rehabilitation programs.

The shoulder complex is composed of three bones (humerus, scapula and clavicle) and four joints as seen in Figure 1. The joints are not very congruent, and thus the shoulder complex has got a great degree of mobility, which can compromise its stability.

The aim of this work is to propose a robust approach, which allows the use of a VICON system as a Dynamic 3D Shape Tracking Device (DSTD) -see endnotes. This novel approach is compared with two methods of the literature (Geometrical Method [START_REF] Wu | ISB recommendation on definitions of joint coordinate systems of various joints for the reporting of human joint motion -Part II: shoulder, elbow, wrist and hand[END_REF] and Point Cluster Method [START_REF] Andriacchi | A point cluster method for in vivo motion analysis: applied to a study of the knee kinematics[END_REF]). Our first goal is to emphasize that a shape-based analysis gives rise to divergent conclusions for some motion components when applied to the scapula in the shoulder complex.

This paper is divided into five parts. After this introduction, section 2 presents a state of the art of motion analysis. Following a summary of the acquisition protocol, section 3 gives the main lines of the proposed method based on the Iterative Median Closest Point algorithm (IMCP) [START_REF] Cresson | Performing accurate rigid kinematics measurements from 3D in vivo image sequences through median consensus simultaneous registration[END_REF] for estimating the motion. In section 4, first results are presented and compared with two other methods of the literature. These first results are discussed in section 5.

II. MOTION ANALYSIS

A. Usual tracking systems

Motion studies can be divided into two groups: static or quasi-static acquisitions, and dynamic acquisitions.

The main static acquisitions were carried out with X-ray photogrammetry [START_REF] Högfors | Biomechanical model of the human shoulder -II. The shoulder rhythm[END_REF], with palpation [START_REF] De Groot | The variability of shoulder motions recorded by means of palpation[END_REF] and with open [START_REF] Beaulieu | Glenohumeral relationships during physiological shoulder motion and stress testing: initial experiment with open MR imaging and active imaging-plane registration[END_REF] or closed [START_REF] Rhoad | A new in vivo technique for three-dimensional shoulder kinematics analysis[END_REF] configuration MRI. The main drawback of these methods is the limited number of positions that can be acquired. Thus, they will not be considered in this paper, which will focus on a dynamic realistic study to understand the shoulder movements.

Dynamic studies use opto-electronic markers [START_REF] Rao | Three-dimensional kinematics of wheel-chair propulsion[END_REF] or magnetic sensors [START_REF] Meskers | Calibration of the "Flock of Birds" electromagnetic tracking device and its application in shoulder motion studies[END_REF]. Their main advantage is the possibility to acquire nearly all sorts of motions under dynamic conditions. Unfortunately, the measures are not directly linked to the bones movement but to the skin deformation. Consequently, some disadvantages can be underlined:

• Skin deformation causes relative displacements between the markers and the underlying bone. Matsui [START_REF] Matsui | Deviation of skin marker from bone target during movement of the scapula[END_REF] measured skin movement errors up to 85 mm for the scapula. In addition, the error value is linked to the nature and range of the motion. • The placement of landmarks depends on the manipulator. Williams [START_REF] Williams | Some aspects of the biomechanics of the elbow joint related to prosthetic design[END_REF] reported mean intra and inter-observer placement errors of about 15 mm.

Shoulder motion analysis using simultaneous skin shape registration

C. Schwartz, Student, M. Lempereur, V. Burdin, Member, J.J. Jacq, Member, O. Rémy-Néris, IEEE T

In spite of good spatial and temporal accuracy, the skin movement errors need an information processing. Several approaches have been developed to correct the marker positions and so, to reduce the influence of skin movements and deformations [START_REF] Leardini | Human movement analysis using stereophotogrammetry Part 3. Soft tissue artefact assessment and compensation[END_REF].

B. Data processing

Veldpaus [START_REF] Veldpaus | A leastsquares algorithm for the equiform transformation from spatial coordinates[END_REF] and Soderkvist [START_REF] Soderkvist | Determining the movements of the skeleton using well-configured markers[END_REF] proposed to minimize the difference between the measured and ideal positions of these markers. Another method, proposed by Chèze [START_REF] Chèze | A solidification procedure to facilitate kinematic analyses based on video system data[END_REF] and named "solidification" computes the movement of the solid, which is the most compatible with the marker trajectories. The solid corresponds to three markers which best represent the bone segment. Proposed by Lu [START_REF] Lu | Bone position estimation from skin marker co-ordinates using global optimisation with joint constraints[END_REF] for the lower limb and applied by Roux [START_REF] Roux | Evaluation of the global optimisation method within the upper limb kinematics analysis[END_REF] to the upper limb, the global optimization method corrects the orientation of the bone whenever relevant joint constraints are a priori available.

In order to correct the skin deformation, other approaches are based on the use of marker cluster rather than individual markers. In this paper, we will compare our new approach with the Point Cluster Method (PCM), proposed by Andriacchi [2] [18] for the lower limb. The PCM attempts to minimize the skin motion artifacts by an optimal and iterative weighting of the markers according to their degree of deformation. The "weight" of each marker is evaluated in order to minimize the variation of the eigenvalues of the inertia matrix of the marker cluster.

III. MATERIALS AND METHODS

A. Protocol

In this study, the shoulder motion is measured with a VICON opto-electronic system composed of seven infrared video cameras MX13 (1.3 MPixels). The acquisition frequency is equal to 120 Hz. In order to compare the different methods, we simultaneously place the markers for each of them in the same acquisition. Skeletal landmarks are 14 mm markers whereas 4 mm markers are used for clusters.

The subject performs five flexions of the arm (in sagittal plane) from rest position to approximately 150° extension. The subject lies flat on his belly. The lying position was chosen for later comparison with MRI acquisitions -this work is not presented in this paper.

The standard geometrical method uses skeletal landmarks. It involves markers positioned following the International Society of Biomechanics (ISB) recommendations [START_REF] Wu | ISB recommendation on definitions of joint coordinate systems of various joints for the reporting of human joint motion -Part II: shoulder, elbow, wrist and hand[END_REF]. Three markers are needed to create a coordinate system. The ISB proposed three skeletal landmarks for the scapula and four others for the thorax (Table 1 and Figure 2). AA, TS and AI are the chosen bone landmarks of the scapula for this study [START_REF] Wu | ISB recommendation on definitions of joint coordinate systems of various joints for the reporting of human joint motion -Part II: shoulder, elbow, wrist and hand[END_REF]. The ISB method uses these three markers to create a local coordinate system on the scapula. The registration of this system is a basic geometric way to estimate its motion [START_REF] Schwartz | Méthode d'analyse du mouvement par utilisation de nappes de marqueurs[END_REF].

While operating through this usual protocol, gaining access to a large marker cluster becomes the main requirement of the IMCP algorithm. Within our first experiments, a cluster of 123 markers is used in order to cover the entire surface of the consecutive locations of the scapula. To our knowledge, such a large cluster has not been reported yet in the literature.

B. Motion estimation from dynamics of the skin shape

As stated before, the main idea is to focus on the instantaneous skin shape. Thus, the available marker cluster must be modeled as a rough point sampling of its embedding continuous surface. The primary information becomes the shape morphology. Upon noise cleanup, the raw marker cluster can be seen as describing the seeding nodes of a 2D parameterization of the skin shape. Thus, the marker locations are no more taken into account as the primary measurements. This relaxes the requirement for a marker to account for a specific location of the underlying bone along the full movement. As this may cancel out the effects of some tangent drifts of its location w.r.t. movement phases, we expect to provide an approach less vulnerable to skin shifts than usual marker-based approaches.

Thus, we are making use of the VICON apparatus as a shape-tracking device -we are not tracking a marker cluster. Conversely, within each time-step, we are focusing on the current polyhedral shape embedding the markers set. As this implies canceling out markers as individual 0D geometric objects, this makes a world of difference. Indeed, the IMCP [START_REF] Jacq | Performing Accurate Joint Kinematics from 3D in vivo Image Sequences through Median Consensus Simultaneous Registration[END_REF][START_REF] Jacq | Performing Accurate Joint Kinematics from 3D in vivo Image Sequences through Consensus-Driven Simultaneous Registration[END_REF][START_REF] Cresson | Performing accurate rigid kinematics measurements from 3D in vivo image sequences through median consensus simultaneous registration[END_REF] approach gives us means to make use of a VICON system as a Dynamic 3D Shape Tracking Device (DSTD). Therefore, we hypothesize that being able to globally and simultaneously track rigid sub-regions of the skin evolving shape will (i) lessen skin-shift-related biases, (ii) give a better accuracy w.r.t. kinematic measurements, (iii) give rise to a better understanding of some insides activities linked to the soft tissues. The previous last point (iii) is related to the availability of the median consensus shape -an intrinsic output of the IMCP algorithm. Indeed, the IMCP is a robust, simultaneous and multi-object extension of the classic algorithm of registration ICP (Iterative Closest Point). The algorithm operates through simultaneous registration of all geometrical instances on a virtual instance accounting for their median consensus. In essence, this method exploits both the spatial and temporal redundancies of the available instance set. Technical descriptions of the IMCP are available elsewhere [START_REF] Jacq | Performing Accurate Joint Kinematics from 3D in vivo Image Sequences through Median Consensus Simultaneous Registration[END_REF][START_REF] Jacq | Performing Accurate Joint Kinematics from 3D in vivo Image Sequences through Consensus-Driven Simultaneous Registration[END_REF]. A short introduction, as well as another application, can be found in [START_REF] Cresson | Performing accurate rigid kinematics measurements from 3D in vivo image sequences through median consensus simultaneous registration[END_REF].

IV. RESULTS

Homogeneous matrices are used to determine the rotational matrix of the scapula with respect to the thorax. Scapular rotations are represented using a standard Euler angle sequence [START_REF] Wu | ISB recommendation on definitions of joint coordinate systems of various joints for the reporting of human joint motion -Part II: shoulder, elbow, wrist and hand[END_REF] of external/internal rotation (Y axis), upward/downward rotation (X axis) and posterior/anterior tilting (Z axis). The distal local coordinate system (Figure 2a) and the proximal local coordinate system (Figure 2b) are created following the ISB recommendations [START_REF] Wu | ISB recommendation on definitions of joint coordinate systems of various joints for the reporting of human joint motion -Part II: shoulder, elbow, wrist and hand[END_REF]. Fig. 2. Skeletal landmarks and coordinate systems of the scapula (Fig. 2a) and of the thorax (Fig. 2b).

Figure 3 depicts the processing of three flexion-extension periods by the IMCP algorithm. In addition to accurate kinematics measurement extracted from the 1200 available shape instances (Fig. 3.d) -whose results are discussed below -the algorithm synthesizes their median consensus shape (Fig. 3.e). This shape is drawn within the lying position depicted by Fig. 3.a. Its color texture aims at underlying high consensus level. This rating-level expresses how-well a region accounts for a rigid-like kinematics along the full sequence. We make the assumption that high consensus regions can be interpreted as fuzzy casts of some underlying bone ridges. As a matter of fact, one can see that two standard ISB anatomical landmarks (TS and AI -see Fig. 2.a) seem to be in good accordance with the consensus level map. However, the main consensus cluster does account for the usual anatomical landmark AA.

The same subject motion was processed by all the three methods. For an easier comparison, the reference posture was defined as the first frame of the motion. Thus, all calculated rotations start from 0°. The results issued from the IMCP are presented in Figure 4. For antero/posterior tilt, the IMCP measures a greater rotation. Measured amplitudes are respectively 22°, 12° and 7° for IMCP, PCM and ISB. For upward/downward and internal/external rotations, all three methods give similar results. Upward/downward rotation mean amplitude is 26° and internal/external mean rotation amplitude is 10°.

V. DISCUSSION

Although an extensive assessment of the measurements is not the primary goal of this first step of our work, one must emphasize here that no ground truth exists in such studies. Thus, in spite of efforts to standardize acquisition procedures, it remains difficult to compare our results with the literature. Indeed, differences in the protocols are not negligible. One of the few validation techniques makes use of pins directly drilled into the scapula. Therefore, this method is not largely widespread since it requires invasive manipulations. Moreover, the interaction of the pins and the muscles may constrain its natural motion. McClure [START_REF] Mcclure | Direct 3-dimentional measurement of scapular kinematics during dynamic movements in vivo[END_REF] uses an electro-magnetic sensor fixed on pins to evaluate the scapula motion. He measures 31° antero/posterior tilt, 46° upward/downward rotation and 26° internal/external rotation. Unfortunately, this work cannot be easily compared to ours. Indeed, McClure's subject was standing whereas ours was lying. Moreover, De Groot [START_REF] De Groot | The variability of shoulder motions recorded by means of palpation[END_REF] shows that intersubject variability of the motion is equal to 5-10°. Meanwhile, the ISB and the PCM seems to under-estimate the real motion of the scapula. Conversely, the IMCP presents encouraging results around the antero/posterior axis. Indeed, the use of a large marker cluster allows covering the scapula from the inferior angle to the acromion. Thus, the IMCP takes takes full advantage of temporal and morphological redundancies. Unlike markers-based approaches (e.g., PCM), it does not consider pieces of data as uncorrelated. It should also be noticed that the few markers on the thorax, which are used to create the proximal coordinate system, also undergo errors due to skin artifacts. This may cause additional errors, when computing the Euler angles from the homogenous matrices.

VI. CONCLUSION

This paper proposes to robustly track the skin shape to recover accurate information about the underlying bone motion. Current applications address the scapula kinematics. The motion estimates are compared to a geometric method (ISB) and a point cluster method (PCM). The results show important differences between the methods. As expected, the IMCP computes greater amplitude. A validation study, by mean of palpations as well as 3D MRI, needs now being carried out in order to corroborate this new but still promising approach. Moreover, a closer examination of the information embedded in the consensus skin shape should bring out new knowledge about soft tissue dynamics.

Endnotes-Here, through the acronym DSTD, we denote a class of markerless device [START_REF] Wu | ISB recommendation on definitions of joint coordinate systems of various joints for the reporting of human joint motion -Part II: shoulder, elbow, wrist and hand[END_REF] able to undergo video sampling rate (i.e., rates better-orequal to 60 Hz) while performing (2) dense (3) regular and (4) simultaneous sampling of an evolving 3D shape with (5) an accuracy compatible with common medical requirements (typ. better-or-equal to 1 mm with distances < 2 m). Our VICON-based hardware emulation mostly fulfills these main requirements (1:120Hz; 3; 4; 5:±1mm). However, for a better adequacy, the markers layer remains to be densified.

Fig. 1 .

 1 Fig. 1. Shoulder complex: bones and joints.

Fig. 3 .

 3 Fig. 3. Processing of three flexion-extension periods by the IMCP algorithm. The data acquisitions convey up to 1200 instances dispatched over 10 sec. Unlike markers-based tracking (b), an interpolating surface (c) is built for each instance locations. Upon simultaneous registrations of the set of surfaces (d), the colors of the median skin shape (e) account for the IMCP consensus level -white means zero consensus (i.e., unreliable parts) whereas red means maxima of the consensus. Views (b) and (d) depicts the whole sweep in thorax coordinate system. The consensus shape (e) is drawn at rest location (t = 0).

Fig. 4

 4 Fig. 4 Rotations of the scapula.relative to the thorax Fig. 4a: Internal/External rotation. Fig. 4b: Anterior/Posterior tilt. Fig. 4c: Upward/Downward rotation.

  Manuscript receivedApril 16, 2007 -revised June 19, 2007. This work was supported by a grant of the Brittany Region.

The authors are with INSERM, U650, Brest, F-29200 France (e-mail: cedric.schwartz@enst-bretagne.fr).

C. Schwartz, J.J. Jacq and V. Burdin are also with GET/ENST Bretagne, Brest, F-29200 France.

C.Schwartz and M.Lempereur are also with Université de Bretagne Occidentale, Brest, F-29200 France O. Rémy-Néris is also with CHU Brest, Service de médecine physique et de réadaptation, Brest, F-29200 France.

Table 1 .

 1 Skeletal landmarks of the thorax and the scapula.

	Bone segments		Skeletal landmarks
		TS	Trigonum Spinae Scapulae
	Scapula	AI	Angulus Inferior
		AA Angulus Acromialis
		C7	Processus Spinosus of the 7 th cervical
			vertebra
	Thorax	T8	Processus Spinosus of the 8 th thoracic vertebra
		IJ	Deepest point of Incisura Jugularis
		PX Processus Xiphoideux