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Towards a passive limitation of particle surface contamination in the Columbus module (ISS) during the MATISS experiment of the Proxima Mission

Towards a passive limitation of particle surface contamination in the Columbus module (ISS)... www.nature.com/npjmgrav ARTICLE OPEN Towards a passive limitation of particle surface contamination in the Columbus module (ISS) during the MATISS experiment of the Proxima Mission 1 2 1 3 4 5 Laurence Lemelle , Lucie Campagnolo , Eléonore Mottin , Denis Le Tourneau , Emmanuel Garre , Pierre Marcoux , 2 2 6 4 5 3 Cécile Thévenot , Alain Maillet , Sébastien Barde , Jérémie Teisseire , Guillaume Nonglaton and Christophe Place Future long-duration human spaceflight calls for developments to limit biocontamination of the surface habitats. The MATISS experiment tests surface treatments in the ISS’s atmosphere. Four sample holders were mounted with glass lamella with hydrophobic coatings, and exposed in the Columbus module for ~6 months. About 7800 particles were detected by tile scanning optical microscopy (×3 and ×30 magnification) indicating a relatively clean environment (a few particles per mm ), but leading to a 2 2 significant coverage-rate (>2% in 20 years). Varied shapes were displayed in the coarse (50–1500 µm ) and fine (0.5–50 µm ) area fractions, consistent with scale dices (tissue or skin) and microbial cells, respectively. The 200–900 µm fraction of the coarse particles was systematically higher on FDTS and SiOCH than on Parylene, while the opposite was observed for the <10 µm fraction of the fine particles. This trend suggests two biocontamination sources and a surface deposition impacted by hydrophobic coatings. npj Microgravity (2020) 6:29 ; https://doi.org/10.1038/s41526-020-00120-w INTRODUCTION that reduce microbial growth and spread on surfaces is a natural next step for new spacecraft generation for longer duration International space agencies plan to advance human spaceflight exploration . through a continued presence in low-Earth orbit (LEO), human Environmental solid surfaces increase the survival ability and missions to cis-lunar space and the lunar surface, and missions to 1,2 infectiosity of microorganisms by their role as sources of nutrients Mars . In this context, the hazardous risks incurred by the and holders that support the development of abundant and astronauts and the equipment’s integrity are challenging in 3–5 complex communities. Once in a biofilm, microorganisms are several respects . Microorganisms can develop over long protected from inhospitable environmental variations and from durations some resistance, unknown mutations to the used 6–8 killing by antibiotics and disinfectants. In the human body, they disinfectants and antibiotics, and virulence , while the cabin’s are at the root of persistent and chronic bacterial infections . bio-contamination by the irreducible microflora of the crew is 5,9,10 Biocontaminated surfaces have been assessed to be infection foci unavoidable . Microorganisms that do not represent severe and transmission routes of pathogens by indirect contact in health hazards for healthy people may become a risk for 23–26 healthcare settings . Biofilm growth in spacecraft under astronauts due to the dysregulation of their immune function . microgravity has been observed experimentally and established On board, air and water are transmission routes of pathogens that to be favorable compared with ground controls, with notable are controlled by filtration systems and monitored both on ground increases of the number of viable cells, biomass, and thickness . and in-flight by microbial analyses. Surfaces also constitute a Metabolic fungal activities on MIR and in the early days of ISS were source of microorganisms which abundances and diversity are 12–15 also identified to be at the origin of equipment degradation by highly variable and controlled . Currently, this particular risk is 29–31 mitigated by a cleaning strategy that consists of manually wiping corrosion . the surface with disinfectants. Besides being time-consuming and Several strategies can be conceived to limit surface bioconta- minations. Bactericidal surfaces have the advantage of killing laborious, it is inefficient for surfaces in inaccessible spaces . This bacteria, but the accumulation of dead bacterial components or risk is increased in spacecraft by longer isolation and a greater extracellular polysaccharides on the surface may paradoxically fuel reliance on an increasing number of closed-loop life support 12,13,16,17 bio-contamination in the long term. Furthermore, evaluating how systems . Furthermore, future long-duration exploration the mechanism of disruption of the metabolic processes of the scenarii will include dormancy periods, where the spacecraft is left microorganism may work on human cells is a considerably unmanned and not sterilized, thus requiring the development of 18,19 autonomous microbial monitoring and control systems . It has expensive pre-requisite. In this respect, surfaces that reduce been discovered that an efficient approach to reduce the risks microbial attachment and repel microorganisms would avoid this associated with microorganisms is to intervene in the design drawback and additionally avoid microorganisms becoming phase of new spacecraft . Choosing materials with surfaces that trapped in the air filtration system. Such surfaces need to be do not contribute to microbial growth and spread is already a designed using few compounds firmly anchored to the surface, prerequisite . Developing sustainable materials and equipment and having as low chemical and nanoparticle toxicities as possible. 1 2 Univ Lyon, ENS de Lyon, Univ Claude Bernard, CNRS, Laboratoire de Géologie de Lyon-Terre Planètes et Environnement, Lyon, France. MEDES-IMPS for CADMOS, Toulouse, 3 4 5 France. Univ Lyon, ENS de Lyon, CNRS, Laboratoire de Physique, Lyon, France. Surface du Verre et Interface, UMR CNRS/Saint-Gobain, Aubervilliers, France. Université Grenoble Alpes, CEA, LETI, DTBS, Grenoble, France. CNES, Toulouse, France. email: laurence.lemelle@ens-lyon.fr; christophe.place@ens-lyon.fr Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; L. Lemelle et al. Fig. 1 The MATISS sample holder. a Exploded-view drawing of the lamella holder showing the following top-down series: the polycarbonate lid (in light blue), the aluminum grid (in grey) ensuring the air circulation in a 2 mm-thick interspace on the side of the lid and the encasing and exposure of the lamella to the air on the other side. Kapton seals were fixed on both sides of the lamella to avoid any direct contact with the lamella. A Vitton plate was then adjoined to the aluminum mounting base with two slots to plug Velcro bands. b Photograph of the sample holder (8.5 cm × 6cm × 1.2 cm) before installation on the Return Grid Sensor Housing in the port-side cone of the Columbus module of the ISS. Photograph courtesy of NASA/ESA permissible to use within the public domain. As microorganisms are most likely transported under microgravity clean glass (see “Materials and methods”). The airborne particles in droplets of hydrous solutions and because self-cleaning may be that contaminated the exposed surfaces were collected using the further implemented, hydrophobic coatings are considered an MATISS sample holder (Fig. 1). This sample holder was designed effective first-line . The strategy here is not to reduce the with three aims: high operability and limited need of crew time, a strength of adhesion on a surface of a microorganism suspended safe long-term and unattended exposure of glass surfaces to the in a fluid, which has been documented to involve multiple ISS atmosphere, and the possibility of running optical imaging of 33,34 electrostatic and hydrophobic forces , which vary over time. By the particles confined within the sample holder (See “Data reducing the contact area of water droplets and floating Availability” section). condensates possibly biocontaminated on surfaces, the hydro- In practice, this sample holder can be considered as a vented phobicity allows for the repulsion of water from the surface and container with a slit between a transparent lid and the glass thereby limits the surface contamination, prior to any species- surfaces, allowing a laminar airflow on the surface of the mounted dependent interaction of a microorganism with a surface. glass lamellae. The transition from the “laboratory-confined” state The Matiss (Microbial Aerosol Tethering on Innovative Surfaces to the “ISS-exposed” state of the glass surfaces was manually in the International Space Station) experiment was designed to operated by removing a Kapton tape that sealed the slit, and investigate if hydrophobic coatings already implemented in reversely by repositioning the Kapton tape. numerous industrial fields could be applied to spacecrafts to limit bio-contamination. During the Matiss experiment, surfaces were The diversity of the surface particles and the potential foci of exposed over long periods of time on the International Space infection Station using a holder designed for this application. Once The size and number of particles collected within the MATISS returned, the exposed surfaces were analyzed by optical micro- sample holder after six months of exposure in the Columbus scopy in the returned, and confined holder and all the particles module were observed across the confined device by tile scanning present on the surfaces were listed. This approach impedes optical microscopy at two different magnifications (Materials and gathering information about the abundances and speciation of methods). the microorganisms present on the surfaces using staining or At low magnification, particles with an area value bigger than swabbing-based techniques. However, by setting sights on the 50 µm were identified. particulate contaminations, this approach provides the possibility The particle size distribution and the corresponding cumulative of tracing further the different sources and routes of the surface curve (Fig. 2a) of 12 lamellae display a monomodal distribution of biocontamination in the ISS. In this study, we report on the 4678 particles with area values in the range of 50–1500 µm with diversity of the particles observed on surfaces exposed for the most probable size equal to ~155 µm . This corresponds to an 6 months in the Columbus module. average density of fewer than two particles per square millimeter −2 (1.6 ± 0.2 particle mm , Supplementary Fig. 1). RESULTS The sharp focus all over the particle area points out their flatness (depth of field » 55 µm in Fig. 2b, Supplementary Fig. 2) Exposure of surfaces to the Columbus atmosphere whatever their elongation ratio. The very flat shape of the particles We report on the diversity of particles collected at two sites with a (thickness < 1 µm) with straight linear sides forming a polygonal low frequency of astronaut contact and good airflow in the shape and an area bigger than 15 µm × 15 µm are consistent with Columbus module, on four types of surface coating that were the desquamated scales of a single or a few keratinized exposed for ~6 months. The fluid dynamics in the environment of 35,36 corneocytes from the astronauts’ epidermises . The origin of the two sites is documented (see “Materials and methods”, the most abundant smaller particles is difficult to ascertain based “Sampling with the MATISS sample holder”) . The air exchange was performed with eight inlets and one large return grid, with a on shape criteria only and they may have been inherited from an flow rate value of ca. 400 m /h, about one order of magnitude inorganic mineral source or have been produced by the partial higher than in ground ventilation, with velocity values of the degradation of the biggest particles. Above the 1500 µm thresh- laminar airflow in the range of 10–40 ft/min near the holders. old (Fig. 2c), fewer than 180 sub-millimeter particles were These surfaces were hydrophobic coated glass surfaces (FDTS, observed, among which one distinct morphotype could be SiOCH, and Parylene) and a hydrophilic surface of the non-treated unambiguously differentiated based on the elongation ratio. npj Microgravity (2020) 29 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; L. Lemelle et al. Fig. 2 Surface contamination by coarse particles. a Particle size distribution histogram (blue bars) with the area in μm and cumulative particle size curve (red) for 4678 particles. b, c Mosaic of optical images recorded at high magnification displaying typical shapes of coarse (left, scale bar is 10 μm) and macroscopic (right, scale bar is 100 μm) particles (area > 1500 μm ). Fig. 3 Surface contamination by fine particles. a Particle size distribution histogram (blue bars) with the area in μm and cumulative particle size curve (red) for 3175 particles b, c Mosaic of optical images displaying single particles with an area smaller than 5 μm² (left, scale bar is 2 μm) and segmented round and elongated particles (right, scale bar is 5 μm). On the longitudinal view of the fibers, they appear to have a The diversity of surface particles on the different hydrophobic ribbon structure irregularly twisted, with a non-circular and coatings irregular diameter of <30 µm and variable lengths. They could The diversity of the surface particles formed on the different seemingly be textile fibers, either cellulosic from clothes, or coatings deposited on the glass lamella distributions was polymeric and fiberglass from the Beta-cloth and the multi-layers investigated. The three coatings FDTS, SiOCH, and Parylene, of which hydrophobicity is related to the water contact angle insulation (MLI) (Supplementary Fig. 3). The largest round and thick particles display complex 3D structures and are therefore decreasing from 110° to 87° (see section Surface coatings in “Materials and methods”), were analyzed. First, the relative difficult to attribute to a specific source of contamination. fractions of the particles compared with the area of the particles At high magnification, particles with an area value as small as were compiled for each type of coating and displayed as a 0.50 µm were observed. The particle size distribution and the cumulative particle size function (Fig. 4). An average function from corresponding cumulative curve for the 4 lamellae (Fig. 3a) three of the exposed sample holders and the corresponding displayed 3175 particles with area values in the range of standard deviation for each of the area fractions, used here as 0.50–50 µm . This corresponds to an average density of about error bars, were evaluated. It was also preliminarily verified that 3.3 particles per square millimeter. the coarse particle density values measured in the three sample About half of the population has an average area value and a holders for each type of coating were quite comparable (standard shape consistent with those of a single cocci (Fig. 3b) while the error lower than 15%, (see Table 1)). The main results are reported others, being either round or elongated (Fig. 3c, Supplementary for the coarse (Fig. 4a) and the fine (Fig. 4b) particles, respectively. Fig. 4), often display constriction figures, consistent with division 2 2 As regards the coarse particles (50 µm <Area< 900 µm )(Table 1), features of filamentous microbial cells or cocci. the fractions of particles with area values lower than 200 µm are We therefore observed two types of contamination on the not significantly different (i.e., within less than one standard exposed surfaces inside the ISS: (i) macroscopic particles that can deviation). The fractions on the glass lamellae coated with FDTS hold microbial cells on their own surface and which number can and SiOCH are systematically higher than the fractions observed probably be restricted by sweep operation and (ii) microscopic on the glass lamellae coated with Parylene for the area values in particles among which several could be remnants of microbial the 200–900 µm range. The difference can be ascribed to a cells deposited and strongly adsorbed onto the surfaces. higher fraction of the smallest particles on FDTS and SIOCH. Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2020) 29 L. Lemelle et al. Fig. 4 Surface contamination and surface treatments. a Cumulative particle size function of coarse particles (50 μm² < Area < 1500 μm )in percentage per Area unit for FDTS (blue), SiOCH (green) and Parylene (red) surface coatings. Counts of particles measured from images recorded at low magnification. Statistical differences between parylen and either FDTS or SiOCH distributions were examined by unpaired Student’s t-test and found to be significantly different (**p-values < 0.01), contrary to that of FDTS and SiOCH (p = 0.4). b Cumulative particle size function of fine particles (0.5 μm² < Area < 50 μm²) in percentage per Area unit for FDTS (blue), SiOCH (green) and Parylene (red) surface coatings. Counts of particles measured from images recorded at high magnification. Error bars represent the standard deviations of the data (see description in the text). Statistical differences between parylen and either FDTS or SiOCH distributions were examined by unpaired Student’s t-test and found to be significantly different (***p-values < 0.001) contrary to that of FDTS and SiOCH (p = 0.13). c Mosaic of optical images displaying a bright halo around single particles (scale bar is 2 μm). particles that could have been formed by the drying of a droplet Table 1. Coarse particle density values, d (particles/mm ), measured in on the surface (Fig. 4c). two sample holders exposed near the Return Grid Sensor Housing (RGHS) and one sample holder on the European Physiology Modules DISCUSSION Facility (EPM) front panel, on three different coatings (FDTS, SiOCH, Parylene). This study establishes experimental proof-of-concept that the MATISS sample holder is useful and adequate for investigating the RGHS RGHS EPM <d> particulate contamination after long-term exposure of surfaces in the ISS habitat once returned to ground. The low density of only a FDTS 1.99 ± 0.55 3.25 ± 0.95 2.12 ± 0.43 2.45 ± 0.64 few particles per mm observed after six months’ exposure in one SiOCH 1.54 ± 0.65 1.39 ± 0.51 1.22 ± 0.24 1.38 ± 0.47 of the dirtiest locations in an instrumental module indicates Parylene 1.37 ± 0.10 1.70 ± 0.33 0.90 ± 0.37 1.33 ± 0.27 relatively clean surfaces, corresponding to a Surface Cleanliness by <d> 1.63 ± 0.32 2.11 ± 0.99 1.41 ± 0.63 Particle concentration of class 6 (>1 µm) . However, if this rate is extrapolated to the foreseen lifetime of a spacecraft’s cabin of several decades, the final coverage of a surface reaches a value higher than 2% (2.2%) in 20 years, which is well above the 9,12,35 Table 2. Fine particle density values, d (particles/mm²), measured in reported safety threshold for electronic equipment . two sample holders exposed near the Return Grid Sensor Housing The diversity of the contaminating particulates on the surfaces (RGHS) and one sample holder on the European Physiology modules displayed in this study results from that of the aerosols that are Facility (EPM) front panel, on three different coatings (FDTS, SiOCH, inherited from the different sources of particles and their Parylene). transportation in the Columbus module. The ISS’s aerosols surveys display a coarse particles fraction specifically formed RGHS RGHS EPM <d> under microgravity due to the absence of the sedimentation of particles >100 µm. Under microgravity, the transportation of FDTS 1.43 8.35 4 ± 1.91 4.45 ± 3.07 aerosols is impacted by the absence of thermal turbulent flow SiOCH 2.18 1.07 5.98 ± 1.94 3.80 ± 2.79 (that is generated on Earth by changes in air density due to heat Parylene 3.45 5.98 2.02 ± 0.49 3.37 ± 1.89 gradients), so in the laminar flows mixing is considerably reduced. <d> 2.36 ± 1.02 5.13 ± 3.72 4 ± 1.98 Only Brownian motion, and electrostatic and phoretic interac- tions allow the motion of the particles and finally their contact with surfaces. Modeling particle deposits, both the fluid dynamics The average particle densities are almost double on the FDTS than computation and the experimental , thus requires an aerosol on SiOCH and Parylene. 2 2 model that is not yet fully updated in low-level activity As regards the fine particles (0.5 µm < Area < 50 µm ) (Table 2), 38,41 environments in particular in the Columbus Module . The the fractions of particles are not significantly different on the glass 3 −1 maximum acceptable values for the airflow are <450 m h and lamellae coated with FDTS or SiOCH (i.e. within less than one for the concentration of particulate matter of the US Environ- standard deviation) (Fig. 4b). These fractions are systematically mental Protection Agency of the National Ambient Air Quality lower than the one observed with Parylene, on which the fractions −3 Standards is 0.05–1mgm of particles <10 μm in aerodynamic of the low-area particles (Area < 10 µm ) is higher. The higher diameter . Considering these values, a maximum flow of 200 mg hydrophobicity of FDTS and SIOCH disfavors contamination by (down to 10 mg) over the glass lamella during the 6 months of small particles probably brought to the surface through water exposure can be estimated. The measured values of particle droplet deposition. Some droplets transportation is indeed density measured herein are several orders of magnitude lower supported by the regular circular halo observed around a few than this corresponding calculated density, indicating particle npj Microgravity (2020) 29 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA L. Lemelle et al. concentrations maintained well below the tolerated threshold FDTS to the surface by a silanisation reaction. The optical thickness of the FDTS layer extrapolated from measurement obtained by Surface-Enhanced value in the Columbus Module and/or reduced fraction of the Ellipsometric Constrast technique on thermally oxidized silicon substrates aerosols deposited on surfaces. was 1.6 ± 0.2 nm. The water contact angle, measured by goniometer Seeing the diversity of the contaminating particulates on the equipment from GBX instruments, on these layers of FDTS was ~110 ± 2°. surfaces displayed here also brings some insights to the potential SiOCH thin films were deposited onto a 200 mm radiofrequency types of foci and transmission routes of pathogens in the capacitive-coupled parallel-plate reactor from Applied Material (using a Columbus Module. A better understanding of them, coupled with plasma excitation frequency at 13.56 MHz). Octamethylcyclotetrasiloxane that of the microbial loads of the different types of particles (OMCTS), obtained from Sigma-Aldrich, was used as precursor, as-received constitute a preliminary step towards modeling surface bioconta- without any further purification. Depositions were performed under mination. Surface microbial (bacterial and fungi) concentrations vacuum (2 Torr) at 100 °C and film thicknesses after deposition were firstly have been reported to fluctuate within a broad range, i.e., from measured by spectroscopic ellipsometry on silicon wafers before deposi- −3 2 12 tion onto glass substrates. The thickness of SiOCH thin films was 1 µm. The 5×10 to 35 CFU/mm , though much lower or comparable to water contact angle, measured by goniometer equipment from GBX the concentrations reported in this study. However, these instruments, on these layers of SiOCH, was ~105 ± 2°. averaged values were evaluated by swabbing the surfaces. They The Parylene layer was deposited using Vapor Deposition System PDS therefore do not take into account the specific microbial loads of 2010 Labcoter® 2 from SCS with Parylene C (Dichloro-di-para-xylylene) as a the different types of particles. This knowledge would be precursor. The deposition conditions for the Parylene layer were as follows. interesting to discover more efficient processes of surface First, dichloro-di-para-xylylene was sublimated at 150 °C under vacuum cleaning. (1 Torr). Then, pyrolysis occurred at 680 °C under vacuum (0.5 Torr). Finally, Practically, manual sweeping is expected to be more efficient the deposition was performed at 25 °C under vacuum (0.1 Torr). The on the largest particles than on the smaller ones that interact thickness of the Parylene layer, measured by spectroscopic ellipsometry, was 5 µm. The water contact angle, measured by goniometer equipment more strongly with the surfaces. The observation of micrometer- from GBX instruments, on these layers of Parylene was ~87 ± 4°. sized particulates made in this study suggests that implementing hydrophobic coatings is an interesting approach to reducing surface biocontamination . In terms of practical implementation Sampling with the MATISS sample holder of coatings for substantially larger areas of spacecraft building MATISS sample holders were mounted with Parylene, FDTS and SiOCH materials, other chemical treatments, such as surface polymeriza- coated surfaces, sealed with Kapton tape, and placed into two Ziploc bags tion deposited by different routes under atmospheric pressure can using gloves. They were brought into the Columbus module by Cygnus CRS OA-5 on 17 October 2016 as part of the MATISS experiment. They were now be considered. mounted by an astronaut in two sites with a low frequency of astronaut The necessity to cope with dormancy periods during unmanned contact and good airflow. Two holders were mounted in the direct vicinity phases, of one to two years for cis-lunar missions, and up to of the Return Grid Sensor Housing (Supplementary Fig. 5A), the most several years for Mars missions, is a critical aspect of near-future important intake of air in the Columbus Cabin. This Grid is located near the human spaceflights . Aside from the need for passive contam- 3 hatch and sucks in air at a rate of 400 m /h. Part of this air is re-injected ination control hardware to contribute to the maintenance of an into the cabin, while the majority is sent to the next module. Air velocity appropriate level of cleanliness of the spacecraft, fully automa- was modeled at 0.21 m/s (∼40 ft/min = 0.21 m/s). Crew activity in this tized devices for microbial monitoring and control procedures location is carried out quickly and limited to maintenance or cleaning while keeping the systems running at their minimum to decrease tasks. One holder was mounted on the surface of the EPM Rack front panel (Supplementary Fig. 5B). This rack is located in the middle section of the energy consumption will be required. A possible strategy to Columbus cabin. The main source of airflow in this location is an air out- realize such sensors might consist of developing an advanced take located ~1 m above the exposure location blowing 55 m /h of air at a MATISS mechanical hardware to probe on the ground not only the speed of 0.21 m/s. The flow is not directed towards the rack surface but number and size of the particles but also some information on towards the center of the cabin. Crew activity around this location is their chemical and biological nature, using only non-invasive but regular. They were exposed to air by removing the Kapton tape on 21 penetrative radiations across the confined setup. This step would November 2016. Sample holders were sealed with Kapton tape using be of particular significance to the better address which gloves, placed into two Ziploc bags and stored at room temperature the technologies should be integrated into the designs of autono- day before the return with the Soyuz 49S on 2 June 2017. Samples were mous and miniaturized sensors to control contamination in situ in transferred on 9 June 2017 to our laboratory, avoiding X-ray scans and dormant spacecraft. A better knowledge of the nature of the ISS’s using temperature monitoring to ensure that no inadvertent extreme temperature events were applied to the sample holders (values in the surface contaminations will be of benefit to their design. range of 5–50 °C). They were stored at 5 °C. METHODS Optical microscopy and image analysis Surface coatings The MATISS sample holder was mounted on a raster X-Y table and a tile For this study, we selected nano particle-free surface coatings deposited scanning mode was applied to image the full glass surface visible across with solvent-free automatized techniques, compatible with a wide range of the polycarbonate cover using the optical macroscope MacroFluo Leica materials, including glass slides, and with hydrophobic surface properties. Z16 ApoA and a PlanApo 5 × /0.5 coupled to a QImaging QICAM fast 1394 camera (12 bits, 1392 × 1040) controlled by a MetaMorph interface. A stack The selected chemical vapor deposition processes were all carried out in a of 30 RGB images (75 ms exposition time) was produced at low zoom (×3), vacuum, which has the advantage in a proof of concept carried out on and of 1452 images (100 ms exposition time) at high zoom (×30). glass lamella to limit the use of potentially toxic organic solvents and to We developed the processing of the stack of images that provided provide an excellent intra and inter lot reproducibility. identifications and optical measurements for each particle with additional FDTS coating is based on (1H,1H,2H,2H)-perfluorodecyltrichlorosilane FDTS (ABCR, 97%). The coating application was performed using data, such as position, area, and elongation ratio. The output was a table commercially available molecular vapor deposition equipment (MVD100 listing each particle found and the features of those particles. For the stack from Applied MST, San José, US). The deposition conditions for FDTS were recorded at low zoom (Supplementary Fig. 6), the segmentation of the as follows. In a first step, the surface was cleaned using remote RF oxygen image was performed on the blue component using a constant threshold plasma (450sccm O flow, 250 W, 300 s). In a second step, one cycle of value of gray level of about 75 that was empirically determined. For the tetrachlorosilane SiCl (Sigma Aldrich, 99,998% Semiconductor grade) at stack recorded at high zoom (Supplementary Fig. 7), the processing of the 18 Torr was injected, followed by four cycles of water at 18 Torr. This step images containing macroscopic objects with shadows masking the small took place for a duration of 600 s at 35 °C. In a third step, two cycles of particles, were removed. The mean intensity of the blue image was FDTS at 0.5 Torr were injected, followed by one cycle of water at 18 Torr. compared to the median value (background) incremented by two times This step took place for a duration of 900 s at 35 °C and aimed at grafting the average value of the standard deviation of the intensity of the stack. Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2020) 29 L. Lemelle et al. The positions of every particle were determined using the Analyze Particle 18. Anderson, M., Sargusingh, M. & Perry, J. Evolution of requirements and module of FIJI. Based on these positions, crops were recorded for each assumptions for future exploration missions. In (eds ICES) Proc. 47th Int. Conf. particle and the segmentation was refined using a local threshold value Environ. Systems,16–20 July (Charleston, South Carolina, 2017). that was a function of the mean value of the crop. At high zoom, crops 19. Pierson, D., Bruce, R., Ott, C. M., Castro, V. & Mehta, S. Microbiological lessons were collected on an 8-bit sum of the RGB images, while high zoom crops learned from the Space Shuttle. In (eds ICES) 41st International Meeting on were sampled on the blue image. The area and the elongation of every Environmental Systems (AIAA, Portland, OR, 2011). particle were then determined using the Analyze Particle module of FIJI. 20. Smirnov, E. M., Ivanov, N. G., Telnov, D. S., Son, C. H. & Aksamentov, V. K. “Computational Fluid Dynamics study of air flow characteristics in the Columbus Module,” SAE Technical Paper 2004-01-2500. https://doi.org/10.4271/2004-01- Reporting summary 2500 (2004). Further information on research design is available in the Nature Research 21. Ott, C. M., Oubre, C., Wallace, S., Mehta, S. & Pierson, D. Risk of adverse health Reporting Summary linked to this article. effects due to host-microorganism interactions. NASA Technical Report JSC-CN- 38050. https://ntrs.nasa.gov/search.jsp?R=20170001973 (2016). 22. Costerton, J. W., Stewart, P. S. & Greenberg, E. P. Bacterial biofilms: a common DATA AVAILABILITY cause of persistent infections. Science 284, 1318–1322 (1999). 23. Otter, J. A., Yezli, S. & French, G. L. The role played by contaminated surfaces in The dataset of particle areas analyzed during the current study are available from the the transmission of nosocomial pathogens. Infect. Control Hosp. Epidemiol. 32, corresponding authors on reasonable request. The MATISS sample holder was 687–699 (2011). deposited at the Institut National de la Propriété. 24. Otter, J. A. et al. Transmission of SARS and MERS coronaviruses and influenza virus in healthcare settings: the possible role of dry surface contamination. J. Hosp. Infect. 92, 235–250 (2016). CODE AVAILABILITY 25. Weber, D., Anderson, D. & Rutala, W. 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International Astronautical Congress (IAF, 2016). npj Microgravity (2020) 29 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA L. Lemelle et al. ACKNOWLEDGEMENTS Correspondence and requests for materials should be addressed to L.L. or C.P. The CNES, the French Space Agency provided financial support for the MATISS Reprints and permission information is available at http://www.nature.com/ hardware design and development as part of the French experiments of the Proxima reprints Mission. Anne-Dominique Malinge and Philippe Bioulez contributed to the qualification of MATISS hardware for space flights and to its final integration. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims Thomas Pesquet, the French European astronaut carried out the MATISS experiments in published maps and institutional affiliations. during his mission. AUTHOR CONTRIBUTIONS L.L., C.P., L.C., A.M., and S.B. designed the research project; D.L. and E.M. machined the Open Access This article is licensed under a Creative Commons sample holder and carried out the microscopy imaging, G.N., P.M., J.T. and E.G. Attribution 4.0 International License, which permits use, sharing, prepared the surfaces, L.C., C.T., A.M., and S.B. managed the qualification for space adaptation, distribution and reproduction in any medium or format, as long as you give flight of the experiment and the communication with the astronaut Th. Pesquet; L.L., appropriate credit to the original author(s) and the source, provide a link to the Creative C.P., and G.N. wrote the paper. Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. 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Towards a passive limitation of particle surface contamination in the Columbus module (ISS) during the MATISS experiment of the Proxima Mission

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www.nature.com/npjmgrav ARTICLE OPEN Towards a passive limitation of particle surface contamination in the Columbus module (ISS) during the MATISS experiment of the Proxima Mission 1 2 1 3 4 5 Laurence Lemelle , Lucie Campagnolo , Eléonore Mottin , Denis Le Tourneau , Emmanuel Garre , Pierre Marcoux , 2 2 6 4 5 3 Cécile Thévenot , Alain Maillet , Sébastien Barde , Jérémie Teisseire , Guillaume Nonglaton and Christophe Place Future long-duration human spaceflight calls for developments to limit biocontamination of the surface habitats. The MATISS experiment tests surface treatments in the ISS’s atmosphere. Four sample holders were mounted with glass lamella with hydrophobic coatings, and exposed in the Columbus module for ~6 months. About 7800 particles were detected by tile scanning optical microscopy (×3 and ×30 magnification) indicating a relatively clean environment (a few particles per mm ), but leading to a 2 2 significant coverage-rate (>2% in 20 years). Varied shapes were displayed in the coarse (50–1500 µm ) and fine (0.5–50 µm ) area fractions, consistent with scale dices (tissue or skin) and microbial cells, respectively. The 200–900 µm fraction of the coarse particles was systematically higher on FDTS and SiOCH than on Parylene, while the opposite was observed for the <10 µm fraction of the fine particles. This trend suggests two biocontamination sources and a surface deposition impacted by hydrophobic coatings. npj Microgravity (2020) 6:29 ; https://doi.org/10.1038/s41526-020-00120-w INTRODUCTION that reduce microbial growth and spread on surfaces is a natural next step for new spacecraft generation for longer duration International space agencies plan to advance human spaceflight exploration . through a continued presence in low-Earth orbit (LEO), human Environmental solid surfaces increase the survival ability and missions to cis-lunar space and the lunar surface, and missions to 1,2 infectiosity of microorganisms by their role as sources of nutrients Mars . In this context, the hazardous risks incurred by the and holders that support the development of abundant and astronauts and the equipment’s integrity are challenging in 3–5 complex communities. Once in a biofilm, microorganisms are several respects . Microorganisms can develop over long protected from inhospitable environmental variations and from durations some resistance, unknown mutations to the used 6–8 killing by antibiotics and disinfectants. In the human body, they disinfectants and antibiotics, and virulence , while the cabin’s are at the root of persistent and chronic bacterial infections . bio-contamination by the irreducible microflora of the crew is 5,9,10 Biocontaminated surfaces have been assessed to be infection foci unavoidable . Microorganisms that do not represent severe and transmission routes of pathogens by indirect contact in health hazards for healthy people may become a risk for 23–26 healthcare settings . Biofilm growth in spacecraft under astronauts due to the dysregulation of their immune function . microgravity has been observed experimentally and established On board, air and water are transmission routes of pathogens that to be favorable compared with ground controls, with notable are controlled by filtration systems and monitored both on ground increases of the number of viable cells, biomass, and thickness . and in-flight by microbial analyses. Surfaces also constitute a Metabolic fungal activities on MIR and in the early days of ISS were source of microorganisms which abundances and diversity are 12–15 also identified to be at the origin of equipment degradation by highly variable and controlled . Currently, this particular risk is 29–31 mitigated by a cleaning strategy that consists of manually wiping corrosion . the surface with disinfectants. Besides being time-consuming and Several strategies can be conceived to limit surface bioconta- minations. Bactericidal surfaces have the advantage of killing laborious, it is inefficient for surfaces in inaccessible spaces . This bacteria, but the accumulation of dead bacterial components or risk is increased in spacecraft by longer isolation and a greater extracellular polysaccharides on the surface may paradoxically fuel reliance on an increasing number of closed-loop life support 12,13,16,17 bio-contamination in the long term. Furthermore, evaluating how systems . Furthermore, future long-duration exploration the mechanism of disruption of the metabolic processes of the scenarii will include dormancy periods, where the spacecraft is left microorganism may work on human cells is a considerably unmanned and not sterilized, thus requiring the development of 18,19 autonomous microbial monitoring and control systems . It has expensive pre-requisite. In this respect, surfaces that reduce been discovered that an efficient approach to reduce the risks microbial attachment and repel microorganisms would avoid this associated with microorganisms is to intervene in the design drawback and additionally avoid microorganisms becoming phase of new spacecraft . Choosing materials with surfaces that trapped in the air filtration system. Such surfaces need to be do not contribute to microbial growth and spread is already a designed using few compounds firmly anchored to the surface, prerequisite . Developing sustainable materials and equipment and having as low chemical and nanoparticle toxicities as possible. 1 2 Univ Lyon, ENS de Lyon, Univ Claude Bernard, CNRS, Laboratoire de Géologie de Lyon-Terre Planètes et Environnement, Lyon, France. MEDES-IMPS for CADMOS, Toulouse, 3 4 5 France. Univ Lyon, ENS de Lyon, CNRS, Laboratoire de Physique, Lyon, France. Surface du Verre et Interface, UMR CNRS/Saint-Gobain, Aubervilliers, France. Université Grenoble Alpes, CEA, LETI, DTBS, Grenoble, France. CNES, Toulouse, France. email: laurence.lemelle@ens-lyon.fr; christophe.place@ens-lyon.fr Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; L. Lemelle et al. Fig. 1 The MATISS sample holder. a Exploded-view drawing of the lamella holder showing the following top-down series: the polycarbonate lid (in light blue), the aluminum grid (in grey) ensuring the air circulation in a 2 mm-thick interspace on the side of the lid and the encasing and exposure of the lamella to the air on the other side. Kapton seals were fixed on both sides of the lamella to avoid any direct contact with the lamella. A Vitton plate was then adjoined to the aluminum mounting base with two slots to plug Velcro bands. b Photograph of the sample holder (8.5 cm × 6cm × 1.2 cm) before installation on the Return Grid Sensor Housing in the port-side cone of the Columbus module of the ISS. Photograph courtesy of NASA/ESA permissible to use within the public domain. As microorganisms are most likely transported under microgravity clean glass (see “Materials and methods”). The airborne particles in droplets of hydrous solutions and because self-cleaning may be that contaminated the exposed surfaces were collected using the further implemented, hydrophobic coatings are considered an MATISS sample holder (Fig. 1). This sample holder was designed effective first-line . The strategy here is not to reduce the with three aims: high operability and limited need of crew time, a strength of adhesion on a surface of a microorganism suspended safe long-term and unattended exposure of glass surfaces to the in a fluid, which has been documented to involve multiple ISS atmosphere, and the possibility of running optical imaging of 33,34 electrostatic and hydrophobic forces , which vary over time. By the particles confined within the sample holder (See “Data reducing the contact area of water droplets and floating Availability” section). condensates possibly biocontaminated on surfaces, the hydro- In practice, this sample holder can be considered as a vented phobicity allows for the repulsion of water from the surface and container with a slit between a transparent lid and the glass thereby limits the surface contamination, prior to any species- surfaces, allowing a laminar airflow on the surface of the mounted dependent interaction of a microorganism with a surface. glass lamellae. The transition from the “laboratory-confined” state The Matiss (Microbial Aerosol Tethering on Innovative Surfaces to the “ISS-exposed” state of the glass surfaces was manually in the International Space Station) experiment was designed to operated by removing a Kapton tape that sealed the slit, and investigate if hydrophobic coatings already implemented in reversely by repositioning the Kapton tape. numerous industrial fields could be applied to spacecrafts to limit bio-contamination. During the Matiss experiment, surfaces were The diversity of the surface particles and the potential foci of exposed over long periods of time on the International Space infection Station using a holder designed for this application. Once The size and number of particles collected within the MATISS returned, the exposed surfaces were analyzed by optical micro- sample holder after six months of exposure in the Columbus scopy in the returned, and confined holder and all the particles module were observed across the confined device by tile scanning present on the surfaces were listed. This approach impedes optical microscopy at two different magnifications (Materials and gathering information about the abundances and speciation of methods). the microorganisms present on the surfaces using staining or At low magnification, particles with an area value bigger than swabbing-based techniques. However, by setting sights on the 50 µm were identified. particulate contaminations, this approach provides the possibility The particle size distribution and the corresponding cumulative of tracing further the different sources and routes of the surface curve (Fig. 2a) of 12 lamellae display a monomodal distribution of biocontamination in the ISS. In this study, we report on the 4678 particles with area values in the range of 50–1500 µm with diversity of the particles observed on surfaces exposed for the most probable size equal to ~155 µm . This corresponds to an 6 months in the Columbus module. average density of fewer than two particles per square millimeter −2 (1.6 ± 0.2 particle mm , Supplementary Fig. 1). RESULTS The sharp focus all over the particle area points out their flatness (depth of field » 55 µm in Fig. 2b, Supplementary Fig. 2) Exposure of surfaces to the Columbus atmosphere whatever their elongation ratio. The very flat shape of the particles We report on the diversity of particles collected at two sites with a (thickness < 1 µm) with straight linear sides forming a polygonal low frequency of astronaut contact and good airflow in the shape and an area bigger than 15 µm × 15 µm are consistent with Columbus module, on four types of surface coating that were the desquamated scales of a single or a few keratinized exposed for ~6 months. The fluid dynamics in the environment of 35,36 corneocytes from the astronauts’ epidermises . The origin of the two sites is documented (see “Materials and methods”, the most abundant smaller particles is difficult to ascertain based “Sampling with the MATISS sample holder”) . The air exchange was performed with eight inlets and one large return grid, with a on shape criteria only and they may have been inherited from an flow rate value of ca. 400 m /h, about one order of magnitude inorganic mineral source or have been produced by the partial higher than in ground ventilation, with velocity values of the degradation of the biggest particles. Above the 1500 µm thresh- laminar airflow in the range of 10–40 ft/min near the holders. old (Fig. 2c), fewer than 180 sub-millimeter particles were These surfaces were hydrophobic coated glass surfaces (FDTS, observed, among which one distinct morphotype could be SiOCH, and Parylene) and a hydrophilic surface of the non-treated unambiguously differentiated based on the elongation ratio. npj Microgravity (2020) 29 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; L. Lemelle et al. Fig. 2 Surface contamination by coarse particles. a Particle size distribution histogram (blue bars) with the area in μm and cumulative particle size curve (red) for 4678 particles. b, c Mosaic of optical images recorded at high magnification displaying typical shapes of coarse (left, scale bar is 10 μm) and macroscopic (right, scale bar is 100 μm) particles (area > 1500 μm ). Fig. 3 Surface contamination by fine particles. a Particle size distribution histogram (blue bars) with the area in μm and cumulative particle size curve (red) for 3175 particles b, c Mosaic of optical images displaying single particles with an area smaller than 5 μm² (left, scale bar is 2 μm) and segmented round and elongated particles (right, scale bar is 5 μm). On the longitudinal view of the fibers, they appear to have a The diversity of surface particles on the different hydrophobic ribbon structure irregularly twisted, with a non-circular and coatings irregular diameter of <30 µm and variable lengths. They could The diversity of the surface particles formed on the different seemingly be textile fibers, either cellulosic from clothes, or coatings deposited on the glass lamella distributions was polymeric and fiberglass from the Beta-cloth and the multi-layers investigated. The three coatings FDTS, SiOCH, and Parylene, of which hydrophobicity is related to the water contact angle insulation (MLI) (Supplementary Fig. 3). The largest round and thick particles display complex 3D structures and are therefore decreasing from 110° to 87° (see section Surface coatings in “Materials and methods”), were analyzed. First, the relative difficult to attribute to a specific source of contamination. fractions of the particles compared with the area of the particles At high magnification, particles with an area value as small as were compiled for each type of coating and displayed as a 0.50 µm were observed. The particle size distribution and the cumulative particle size function (Fig. 4). An average function from corresponding cumulative curve for the 4 lamellae (Fig. 3a) three of the exposed sample holders and the corresponding displayed 3175 particles with area values in the range of standard deviation for each of the area fractions, used here as 0.50–50 µm . This corresponds to an average density of about error bars, were evaluated. It was also preliminarily verified that 3.3 particles per square millimeter. the coarse particle density values measured in the three sample About half of the population has an average area value and a holders for each type of coating were quite comparable (standard shape consistent with those of a single cocci (Fig. 3b) while the error lower than 15%, (see Table 1)). The main results are reported others, being either round or elongated (Fig. 3c, Supplementary for the coarse (Fig. 4a) and the fine (Fig. 4b) particles, respectively. Fig. 4), often display constriction figures, consistent with division 2 2 As regards the coarse particles (50 µm <Area< 900 µm )(Table 1), features of filamentous microbial cells or cocci. the fractions of particles with area values lower than 200 µm are We therefore observed two types of contamination on the not significantly different (i.e., within less than one standard exposed surfaces inside the ISS: (i) macroscopic particles that can deviation). The fractions on the glass lamellae coated with FDTS hold microbial cells on their own surface and which number can and SiOCH are systematically higher than the fractions observed probably be restricted by sweep operation and (ii) microscopic on the glass lamellae coated with Parylene for the area values in particles among which several could be remnants of microbial the 200–900 µm range. The difference can be ascribed to a cells deposited and strongly adsorbed onto the surfaces. higher fraction of the smallest particles on FDTS and SIOCH. Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2020) 29 L. Lemelle et al. Fig. 4 Surface contamination and surface treatments. a Cumulative particle size function of coarse particles (50 μm² < Area < 1500 μm )in percentage per Area unit for FDTS (blue), SiOCH (green) and Parylene (red) surface coatings. Counts of particles measured from images recorded at low magnification. Statistical differences between parylen and either FDTS or SiOCH distributions were examined by unpaired Student’s t-test and found to be significantly different (**p-values < 0.01), contrary to that of FDTS and SiOCH (p = 0.4). b Cumulative particle size function of fine particles (0.5 μm² < Area < 50 μm²) in percentage per Area unit for FDTS (blue), SiOCH (green) and Parylene (red) surface coatings. Counts of particles measured from images recorded at high magnification. Error bars represent the standard deviations of the data (see description in the text). Statistical differences between parylen and either FDTS or SiOCH distributions were examined by unpaired Student’s t-test and found to be significantly different (***p-values < 0.001) contrary to that of FDTS and SiOCH (p = 0.13). c Mosaic of optical images displaying a bright halo around single particles (scale bar is 2 μm). particles that could have been formed by the drying of a droplet Table 1. Coarse particle density values, d (particles/mm ), measured in on the surface (Fig. 4c). two sample holders exposed near the Return Grid Sensor Housing (RGHS) and one sample holder on the European Physiology Modules DISCUSSION Facility (EPM) front panel, on three different coatings (FDTS, SiOCH, Parylene). This study establishes experimental proof-of-concept that the MATISS sample holder is useful and adequate for investigating the RGHS RGHS EPM <d> particulate contamination after long-term exposure of surfaces in the ISS habitat once returned to ground. The low density of only a FDTS 1.99 ± 0.55 3.25 ± 0.95 2.12 ± 0.43 2.45 ± 0.64 few particles per mm observed after six months’ exposure in one SiOCH 1.54 ± 0.65 1.39 ± 0.51 1.22 ± 0.24 1.38 ± 0.47 of the dirtiest locations in an instrumental module indicates Parylene 1.37 ± 0.10 1.70 ± 0.33 0.90 ± 0.37 1.33 ± 0.27 relatively clean surfaces, corresponding to a Surface Cleanliness by <d> 1.63 ± 0.32 2.11 ± 0.99 1.41 ± 0.63 Particle concentration of class 6 (>1 µm) . However, if this rate is extrapolated to the foreseen lifetime of a spacecraft’s cabin of several decades, the final coverage of a surface reaches a value higher than 2% (2.2%) in 20 years, which is well above the 9,12,35 Table 2. Fine particle density values, d (particles/mm²), measured in reported safety threshold for electronic equipment . two sample holders exposed near the Return Grid Sensor Housing The diversity of the contaminating particulates on the surfaces (RGHS) and one sample holder on the European Physiology modules displayed in this study results from that of the aerosols that are Facility (EPM) front panel, on three different coatings (FDTS, SiOCH, inherited from the different sources of particles and their Parylene). transportation in the Columbus module. The ISS’s aerosols surveys display a coarse particles fraction specifically formed RGHS RGHS EPM <d> under microgravity due to the absence of the sedimentation of particles >100 µm. Under microgravity, the transportation of FDTS 1.43 8.35 4 ± 1.91 4.45 ± 3.07 aerosols is impacted by the absence of thermal turbulent flow SiOCH 2.18 1.07 5.98 ± 1.94 3.80 ± 2.79 (that is generated on Earth by changes in air density due to heat Parylene 3.45 5.98 2.02 ± 0.49 3.37 ± 1.89 gradients), so in the laminar flows mixing is considerably reduced. <d> 2.36 ± 1.02 5.13 ± 3.72 4 ± 1.98 Only Brownian motion, and electrostatic and phoretic interac- tions allow the motion of the particles and finally their contact with surfaces. Modeling particle deposits, both the fluid dynamics The average particle densities are almost double on the FDTS than computation and the experimental , thus requires an aerosol on SiOCH and Parylene. 2 2 model that is not yet fully updated in low-level activity As regards the fine particles (0.5 µm < Area < 50 µm ) (Table 2), 38,41 environments in particular in the Columbus Module . The the fractions of particles are not significantly different on the glass 3 −1 maximum acceptable values for the airflow are <450 m h and lamellae coated with FDTS or SiOCH (i.e. within less than one for the concentration of particulate matter of the US Environ- standard deviation) (Fig. 4b). These fractions are systematically mental Protection Agency of the National Ambient Air Quality lower than the one observed with Parylene, on which the fractions −3 Standards is 0.05–1mgm of particles <10 μm in aerodynamic of the low-area particles (Area < 10 µm ) is higher. The higher diameter . Considering these values, a maximum flow of 200 mg hydrophobicity of FDTS and SIOCH disfavors contamination by (down to 10 mg) over the glass lamella during the 6 months of small particles probably brought to the surface through water exposure can be estimated. The measured values of particle droplet deposition. Some droplets transportation is indeed density measured herein are several orders of magnitude lower supported by the regular circular halo observed around a few than this corresponding calculated density, indicating particle npj Microgravity (2020) 29 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA L. Lemelle et al. concentrations maintained well below the tolerated threshold FDTS to the surface by a silanisation reaction. The optical thickness of the FDTS layer extrapolated from measurement obtained by Surface-Enhanced value in the Columbus Module and/or reduced fraction of the Ellipsometric Constrast technique on thermally oxidized silicon substrates aerosols deposited on surfaces. was 1.6 ± 0.2 nm. The water contact angle, measured by goniometer Seeing the diversity of the contaminating particulates on the equipment from GBX instruments, on these layers of FDTS was ~110 ± 2°. surfaces displayed here also brings some insights to the potential SiOCH thin films were deposited onto a 200 mm radiofrequency types of foci and transmission routes of pathogens in the capacitive-coupled parallel-plate reactor from Applied Material (using a Columbus Module. A better understanding of them, coupled with plasma excitation frequency at 13.56 MHz). Octamethylcyclotetrasiloxane that of the microbial loads of the different types of particles (OMCTS), obtained from Sigma-Aldrich, was used as precursor, as-received constitute a preliminary step towards modeling surface bioconta- without any further purification. Depositions were performed under mination. Surface microbial (bacterial and fungi) concentrations vacuum (2 Torr) at 100 °C and film thicknesses after deposition were firstly have been reported to fluctuate within a broad range, i.e., from measured by spectroscopic ellipsometry on silicon wafers before deposi- −3 2 12 tion onto glass substrates. The thickness of SiOCH thin films was 1 µm. The 5×10 to 35 CFU/mm , though much lower or comparable to water contact angle, measured by goniometer equipment from GBX the concentrations reported in this study. However, these instruments, on these layers of SiOCH, was ~105 ± 2°. averaged values were evaluated by swabbing the surfaces. They The Parylene layer was deposited using Vapor Deposition System PDS therefore do not take into account the specific microbial loads of 2010 Labcoter® 2 from SCS with Parylene C (Dichloro-di-para-xylylene) as a the different types of particles. This knowledge would be precursor. The deposition conditions for the Parylene layer were as follows. interesting to discover more efficient processes of surface First, dichloro-di-para-xylylene was sublimated at 150 °C under vacuum cleaning. (1 Torr). Then, pyrolysis occurred at 680 °C under vacuum (0.5 Torr). Finally, Practically, manual sweeping is expected to be more efficient the deposition was performed at 25 °C under vacuum (0.1 Torr). The on the largest particles than on the smaller ones that interact thickness of the Parylene layer, measured by spectroscopic ellipsometry, was 5 µm. The water contact angle, measured by goniometer equipment more strongly with the surfaces. The observation of micrometer- from GBX instruments, on these layers of Parylene was ~87 ± 4°. sized particulates made in this study suggests that implementing hydrophobic coatings is an interesting approach to reducing surface biocontamination . In terms of practical implementation Sampling with the MATISS sample holder of coatings for substantially larger areas of spacecraft building MATISS sample holders were mounted with Parylene, FDTS and SiOCH materials, other chemical treatments, such as surface polymeriza- coated surfaces, sealed with Kapton tape, and placed into two Ziploc bags tion deposited by different routes under atmospheric pressure can using gloves. They were brought into the Columbus module by Cygnus CRS OA-5 on 17 October 2016 as part of the MATISS experiment. They were now be considered. mounted by an astronaut in two sites with a low frequency of astronaut The necessity to cope with dormancy periods during unmanned contact and good airflow. Two holders were mounted in the direct vicinity phases, of one to two years for cis-lunar missions, and up to of the Return Grid Sensor Housing (Supplementary Fig. 5A), the most several years for Mars missions, is a critical aspect of near-future important intake of air in the Columbus Cabin. This Grid is located near the human spaceflights . Aside from the need for passive contam- 3 hatch and sucks in air at a rate of 400 m /h. Part of this air is re-injected ination control hardware to contribute to the maintenance of an into the cabin, while the majority is sent to the next module. Air velocity appropriate level of cleanliness of the spacecraft, fully automa- was modeled at 0.21 m/s (∼40 ft/min = 0.21 m/s). Crew activity in this tized devices for microbial monitoring and control procedures location is carried out quickly and limited to maintenance or cleaning while keeping the systems running at their minimum to decrease tasks. One holder was mounted on the surface of the EPM Rack front panel (Supplementary Fig. 5B). This rack is located in the middle section of the energy consumption will be required. A possible strategy to Columbus cabin. The main source of airflow in this location is an air out- realize such sensors might consist of developing an advanced take located ~1 m above the exposure location blowing 55 m /h of air at a MATISS mechanical hardware to probe on the ground not only the speed of 0.21 m/s. The flow is not directed towards the rack surface but number and size of the particles but also some information on towards the center of the cabin. Crew activity around this location is their chemical and biological nature, using only non-invasive but regular. They were exposed to air by removing the Kapton tape on 21 penetrative radiations across the confined setup. This step would November 2016. Sample holders were sealed with Kapton tape using be of particular significance to the better address which gloves, placed into two Ziploc bags and stored at room temperature the technologies should be integrated into the designs of autono- day before the return with the Soyuz 49S on 2 June 2017. Samples were mous and miniaturized sensors to control contamination in situ in transferred on 9 June 2017 to our laboratory, avoiding X-ray scans and dormant spacecraft. A better knowledge of the nature of the ISS’s using temperature monitoring to ensure that no inadvertent extreme temperature events were applied to the sample holders (values in the surface contaminations will be of benefit to their design. range of 5–50 °C). They were stored at 5 °C. METHODS Optical microscopy and image analysis Surface coatings The MATISS sample holder was mounted on a raster X-Y table and a tile For this study, we selected nano particle-free surface coatings deposited scanning mode was applied to image the full glass surface visible across with solvent-free automatized techniques, compatible with a wide range of the polycarbonate cover using the optical macroscope MacroFluo Leica materials, including glass slides, and with hydrophobic surface properties. Z16 ApoA and a PlanApo 5 × /0.5 coupled to a QImaging QICAM fast 1394 camera (12 bits, 1392 × 1040) controlled by a MetaMorph interface. A stack The selected chemical vapor deposition processes were all carried out in a of 30 RGB images (75 ms exposition time) was produced at low zoom (×3), vacuum, which has the advantage in a proof of concept carried out on and of 1452 images (100 ms exposition time) at high zoom (×30). glass lamella to limit the use of potentially toxic organic solvents and to We developed the processing of the stack of images that provided provide an excellent intra and inter lot reproducibility. identifications and optical measurements for each particle with additional FDTS coating is based on (1H,1H,2H,2H)-perfluorodecyltrichlorosilane FDTS (ABCR, 97%). The coating application was performed using data, such as position, area, and elongation ratio. The output was a table commercially available molecular vapor deposition equipment (MVD100 listing each particle found and the features of those particles. For the stack from Applied MST, San José, US). The deposition conditions for FDTS were recorded at low zoom (Supplementary Fig. 6), the segmentation of the as follows. In a first step, the surface was cleaned using remote RF oxygen image was performed on the blue component using a constant threshold plasma (450sccm O flow, 250 W, 300 s). In a second step, one cycle of value of gray level of about 75 that was empirically determined. For the tetrachlorosilane SiCl (Sigma Aldrich, 99,998% Semiconductor grade) at stack recorded at high zoom (Supplementary Fig. 7), the processing of the 18 Torr was injected, followed by four cycles of water at 18 Torr. This step images containing macroscopic objects with shadows masking the small took place for a duration of 600 s at 35 °C. In a third step, two cycles of particles, were removed. The mean intensity of the blue image was FDTS at 0.5 Torr were injected, followed by one cycle of water at 18 Torr. compared to the median value (background) incremented by two times This step took place for a duration of 900 s at 35 °C and aimed at grafting the average value of the standard deviation of the intensity of the stack. Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2020) 29 L. Lemelle et al. The positions of every particle were determined using the Analyze Particle 18. Anderson, M., Sargusingh, M. & Perry, J. Evolution of requirements and module of FIJI. Based on these positions, crops were recorded for each assumptions for future exploration missions. In (eds ICES) Proc. 47th Int. Conf. particle and the segmentation was refined using a local threshold value Environ. Systems,16–20 July (Charleston, South Carolina, 2017). that was a function of the mean value of the crop. At high zoom, crops 19. 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International Astronautical Congress (IAF, 2016). npj Microgravity (2020) 29 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA L. Lemelle et al. ACKNOWLEDGEMENTS Correspondence and requests for materials should be addressed to L.L. or C.P. The CNES, the French Space Agency provided financial support for the MATISS Reprints and permission information is available at http://www.nature.com/ hardware design and development as part of the French experiments of the Proxima reprints Mission. Anne-Dominique Malinge and Philippe Bioulez contributed to the qualification of MATISS hardware for space flights and to its final integration. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims Thomas Pesquet, the French European astronaut carried out the MATISS experiments in published maps and institutional affiliations. during his mission. AUTHOR CONTRIBUTIONS L.L., C.P., L.C., A.M., and S.B. designed the research project; D.L. and E.M. machined the Open Access This article is licensed under a Creative Commons sample holder and carried out the microscopy imaging, G.N., P.M., J.T. and E.G. Attribution 4.0 International License, which permits use, sharing, prepared the surfaces, L.C., C.T., A.M., and S.B. managed the qualification for space adaptation, distribution and reproduction in any medium or format, as long as you give flight of the experiment and the communication with the astronaut Th. Pesquet; L.L., appropriate credit to the original author(s) and the source, provide a link to the Creative C.P., and G.N. wrote the paper. Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the COMPETING INTERESTS article’s Creative Commons license and your intended use is not permitted by statutory The authors declare no competing interests. regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. ADDITIONAL INFORMATION Supplementary information is available for this paper at https://doi.org/10.1038/ © The Author(s) 2020 s41526-020-00120-w. Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2020) 29

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