Operations of a spaceflight experiment to investigate plant tropisms

Plants will be an important component in bioregenerative systems for long-term missions to the Moon and Mars. Since gravity is reduced both on the Moon and Mars, studies that identify the basic mechanisms of plant growth and development in altered gravity are required to ensure successful plant production on these space colonization missions. To address these issues, we have developed a project on the International Space Station (ISS) to study the interaction between gravitropism and phototropism in Arabidopsis thaliana. These experiments were termed TROPI (for tropisms) and were performed on the European Modular Cultivation System (EMCS) in 2006. In this paper, we provide an operational summary of TROPI and preliminary results on studies of tropistic curvature of seedlings grown in space. Seed germination in TROPI was lower compared to previous space experiments, and this was likely due to extended storage in hardware for up to 8 months. Video downlinks provided an important quality check on the automated experimental time line that also was monitored with telemetry. Good quality images of seedlings were obtained, but the use of analog video tapes resulted in delays in image processing and analysis procedures. Seedlings that germinated exhibited robust phototropic curvature. Frozen plant samples were returned on three space shuttle missions, and improvements in cold stowage and handing procedures in the second and third missions resulted in quality RNA extracted from the seedlings that was used in subsequent microarray analyses. While the TROPI experiment had technical and logistical difficulties, most of the procedures worked well due to refinement during the project.     

Improvements in the re-flight of spaceflight experiments on plant tropisms

In order to effectively study phototropism, the directed growth in response to light, we performed a series of experiments in microgravity to better understand light response without the “complications” of a 1-g stimulus. These experiments were named TROPI (for tropisms) and were performed on the European Modular Cultivation System (EMCS), a laboratory facility on the International Space Station (ISS). TROPI-1 was performed in 2006, and while it was a successful experiment, there were a number of technical difficulties. We had the opportunity to perform TROPI-2 in 2010 and were able to optimize experimental conditions as well as to extend the studies of phototropism to fractional gravity created by the EMCS centrifuge. This paper focuses on how the technical improvements in TROPI-2 allowed for a better experiment with increased scientific return. Major modifications in TROPI-2 compared to TROPI-1 included the use of spaceflight hardware that was off-gassed for a longer period and reduced seed storage (less than 2 months) in hardware. These changes resulted in increased seed germination and more vigorous growth of seedlings. While phototropism in response to red illumination was observed in hypocotyls of seedlings grown in microgravity during TROPI-1, there was a greater magnitude of red-light-based phototropic curvature in TROPI-2. Direct downlinking of digital images from the ISS in TROPI-2, rather than the use of analog tapes in TROPI-1, resulted in better quality images and simplified data analyses. In TROPI-2, improved cryo-procedures and the use of the GLACIER freezer during transport of samples back to Earth maintained the low temperature necessary to obtain good-quality RNA required for use in gene profiling studies.     

The performance of KSC Fixation Tubes with RNALater for orbital experiments: A case study in ISS operations for molecular biology

Molecular biology experiments on the International Space Station (ISS) continue to face challenges of sample harvesting and sample return to earth for post flight analysis; however, the use of Kennedy Space Center Fixation Tubes filled with RNALater has proven to be a robust solution to many of these challenges. While it is clear that one direction of future spaceflight experimentation may be towards enhanced on-orbit analytical capabilities, the rapid progress of earth-bound analytical capacity dictates that facile return of molecular biology samples from the ISS will continue to be a mainstay of space life sciences research and flight operations. In this paper we present a case study of the successful performance of KFTs and RNALater over a broad set of operational conditions of ascent configuration, on-orbit experiment use, on-orbit storage and sample return configurations that are unique to ISS current operations and constraints. We also provide observations on performance limits and discuss deployment opportunities and scenarios that are consistent with continued successful ISS molecular biology experimentation.     

Simulations of absorbed dose on the phantom surface of MATROSHKA-R experiment at the ISS

The health risks associated with exposure to various components of space radiation are of great concern when planning manned long-term interplanetary missions, such as future missions to Mars. Since it is not possible to measure the radiation environment inside of human organs in deep space, simulations based on radiation transport/interaction codes coupled to phantoms of tissue equivalent materials are used. However, the calculated results depend on the models used in the codes, and it is therefore necessary to verify their validity by comparison with measured data. The goal of this paper is to compare absorbed doses obtained in the MATROSHKA-R experiment performed at the International Space Station (ISS) with simulations performed with the three-dimensional Monte Carlo Particle and Heavy-Ion Transport code System (PHITS). The absorbed dose was measured using passive detectors (packages of thermoluminescent and plastic nuclear track detectors) placed on the surface of the spherical tissue equivalent phantom MATROSHKA-R, which was exposed aboard the ISS in the Service Zvezda Module from December 2005 to September 2006. The data calculated by PHITS assuming an ISS shielding of 3 g/cm² and 5 g/cm² aluminum mass thickness were in good agreement with the measurements. Using a simplified geometrical model of the ISS, the influence of variations in altitude and wall mass thickness of the ISS on the calculated absorbed dose was estimated. The uncertainties of the calculated data are also discussed; the relative expanded uncertainty of absorbed dose in phantom was estimated to be 44% at a 95% confidence level.     

Radiation measurements inside a human phantom aboard the International Space Station using Liulin-5 charged particle telescope

Liulin-5 is a particle telescope developed for the investigation of the radiation environment within the Russian spherical tissue-equivalent phantom on the International Space Station (ISS). Liulin-5 experiment is conducted aboard the Russian segment of ISS since 28 June 2007 as an adherent part of the international project MATROSHKA-R. The main objective of Liulin-5 experiment is to study the depth-dose distribution of the different components of the orbital radiation field in a human phantom. Additional objectives are mapping of the radiation environment in the phantom and its variations with time and orbital parameters (such as solar cycle, solar flare events, inclination and altitude). Liulin-5 is an active instrument, capable to provide real-time radiation data for the particle flux and dose rates, energy deposition and LET spectra. Data are recorded automatically on memory cards, periodically transported to ground by returning vehicles. In this report we present some first results from data analysis including energy deposition spectra, absorbed dose, dose rate and flux distribution measured simultaneously at 3 different depths of phantom’s radial channel and linear energy transfer (LET) spectrum. Data discussed are for the period July 2007–April 2008.     

Life sciences flight hardware development for the International Space Station.

During the construction phase of the International Space Station (ISS), early flight opportunities have been identified (including designated Utilization Flights, UF) on which early science experiments may be performed. The focus of NASA's and other agencies' biological studies on the early flight opportunities is cell and molecular biology; with UF-1 scheduled to fly in fall 2001, followed by flights 8A and UF-3. Specific hardware is being developed to verify design concepts, e.g., the Avian Development Facility for incubation of small eggs and the Biomass Production System for plant cultivation. Other hardware concepts will utilize those early research opportunities onboard the ISS, e.g., an Incubator for sample cultivation, the European Modular Cultivation System for research with small plant systems, an Insect Habitat for support of insect species. Following the first Utilization Flights, additional equipment will be transported to the ISS to expand research opportunities and capabilities, e.g., a Cell Culture Unit, the Advanced Animal Habitat for rodents, an Aquatic Facility to support small fish and aquatic specimens, a Plant Research Unit for plant cultivation, and a specialized Egg Incubator for developmental biology studies. Host systems (Figure 1A, B: see text), e.g., a 2.5 m Centrifuge Rotor (g-levels from 0.01-g to 2-g) for direct comparisons between g and selectable g levels, the Life Sciences Glovebox for contained manipulations, and Habitat Holding Racks (Figure 1B: see text) will provide electrical power, communication links, and cooling to the habitats. Habitats will provide food, water, light, air and waste management as well as humidity and temperature control for a variety of research organisms. Operators on Earth and the crew on the ISS will be able to send commands to the laboratory equipment to monitor and control the environmental and experimental parameters inside specific habitats. Common laboratory equipment such as microscopes, cryo freezers, radiation dosimeters, and mass measurement devices are also currently in design stages by NASA and the ISS international partners.     

3D information from 2D images recorded in the European Modular Cultivation System on the ISS

The European Modular Cultivation System (EMCS) on the ISS allows long-term biological experiments, e.g. on plants. Video cameras provide near real-time 2D images from these experiments. A method to obtain 3D coordinates and stereoscopic images from these 2D images has been developed and is described in this paper. The procedure was developed to enhance the data output of the MULTIGEN-1 experiment in 2007. One of the main objectives of the experiment was to study growth movements of the Arabidopsis plants and the effect of gravity on these. 3D data were important during parts of the experiment and the paper presents the method developed to acquire 3D data, the accuracy of the data, limitations to the technique and ways to improve the accuracy. Sequences of 3D data obtained from the MULTIGEN-1 experiment are used to illustrate the potential of this newfound capability of the EMCS. In the experiment setup, a positional depth accuracy of about ±0.4 mm for relative object distances and an absolute depth accuracy of about ±1.4 mm for time dependent phenomena was reached. The ability to both view biological specimens in 3D as well as obtaining quantitative 3D data added greatly to the scientific output of the MULTIGEN-1 experiment. The uses of the technique to other researchers and their experiments are discussed.     

Science Researches of Chinese Manned Space Flight

With the complete success of the 2nd stage of Chinese Manned Space Program (CMSP), several science researches have been performed on Tiangong-1 experimental spacelab, which was docked with three Shenzhou spaceships one after another. The China's real spacelab, Tiangong-2 will be launched in 2015, docked with a Shenzhou spaceship soon. After six months, it will be docked with the first Chinese cargo ship (Tianzhou-1). More space science researches, involving with space biology, fluid physics, fundamental physics, materials science, Earth science, astronomy and space environmental science, will be operated on Tiangong-2 spacelab, and crewed and cargo spaceships. Furthermore, the considerable large-scale space utilization of Shina's Space Station is planned. The research fields include yet not limited to space medicine and physiology, space life science and biotechnology, fluid physics and combustion in microgravity, space material science, and fundamental physics in microgravity, space astronomy, Earth science, space physics and space environment utilization, technology demonstration.      

Clover development during spaceflight: a model system.

The development of legume root nodules was studied as a model system for the examination of gravitational effects on plant root development. In order to examine whether rhizobial association with clover roots can be achieved in microgravity, experiments were performed aboard the KC-135 parabolic aircraft and aboard the sounding rocket mission Consort 3. Binding of rhizobia to roots and the initial stages of root nodule development successfully occurred in microgravity. Seedling germination experiments were performed in the sliding block device, the Materials Dispersion Apparatus, aboard STS-37. When significant hydration of the seeds was achieved, normal rates of germination and seedling development were observed.     

Simulations of MATROSHKA experiment outside the ISS using PHITS

The radiation environment at the altitude of the International Space Station (ISS) is substantially different than anything typically encountered on Earth in both the character of the radiation field and the significantly higher dose rates. Concerns about the biological effects on humans of this highly complex natural radiation field are increasing due to higher amount of astronauts performing long-duration missions onboard the ISS and especially if looking into planned future manned missions to Mars. In order to begin the process of predicting the dose levels seen by the organs of an astronaut, being the prerequisite for radiation risk calculations, it is necessary to understand the character of the radiation environment both in- and outside of the ISS as well as the relevant contributions from the radiation field to the organ doses.     

Implementation of ALARA radiation protection on the ISS through polyethylene shielding augmentation of the Service Module Crew Quarters.

With 5-7 month long duration missions at 51.6 degrees inclination in Low Earth Orbit, the ionizing radiation levels to which International Space Station (ISS) crewmembers are exposed will be the highest planned occupational exposures in the world. Even with the expectation that regulatory dose limits will not be exceeded during a single tour of duty aboard the ISS, the "as low as reasonably achievable" (ALARA) precept requires that radiological risks be minimized when possible through a dose optimization process. Judicious placement of efficient shielding materials in locations where crewmembers sleep, rest, or work is an important means for implementing ALARA for spaceflight. Polyethylene (CnHn) is a relatively inexpensive, stable, and, with a low atomic number, an effective shielding material that has been certified for use aboard the ISS. Several designs for placement of slabs or walls of polyethylene have been evaluated for radiation exposure reduction in the Crew Quarters (CQ) of the Zvezda (Star) Service Module. Optimization of shield designs relies on accurate characterization of the expected primary and secondary particle environment and modeling of the predicted radiobiological responses of critical organs and tissues. Results of the studies shown herein indicate that 20% or more reduction in equivalent dose to the CQ occupant is achievable. These results suggest that shielding design and risk analysis are necessary measures for reducing long-term radiological risks to ISS inhabitants and for meeting legal ALARA requirements. Verification of shield concepts requires results from specific designs to be compared with onboard dosimetry.     

Growth of pea epicotyl in low magnetic field implication for space research

A magnetic field is an inescapable environmental factor for plants on the earth. However, its impact on plant growth is not well understood. In order to survey how magnetic fields affect plant, Alaska pea seedlings were incubated under low magnetic field (LMF) and also in the normal geo-magnetic environment. Two-day-old etiolated seedlings were incubated in a magnetic shield box and in a control box. Sedimentation of amyloplasts was examined in the epicotyls of seedlings grown under these two conditions. The elongation of epicotyls was promoted by LMF. Elongation was most prominent in the middle part of the epicotyls. Cell elongation and increased osmotic pressure of cell sap were found in the epidermal cells exposed to LMF. When the gravitational environment was 1G, the epicotyls incubated under both LMF and normal geomagnetic field grew straight upward and amyloplasts sedimented similarly. However, under simulated microgravity (clinostat), epicotyl and cell elongation was promoted. Furthermore, the epicotyls bent and amyloplasts were dispersed in the cells in simulated microgravity. The dispersion of amyloplasts may relate to the posture control in epicotyl growth under simulated microgravity generated by 3D clinorotation, since it was not observed under LMF in 1G. Since enhanced elongation of cells was commonly seen both at LMF and in simulated microgravity, all elongation on the 3D-clinostat could result from pseudo-low magnetic field, as a by-product of clinorotation. (i.e., clinostat results could be based on randomization of magnetic field together with randomization of gravity vector.) Our results point to the possible use of space for studies in magnetic biology. With space experiments, the effects of dominant environmental factors, such as gravity on plants, could be neutralized or controlled for to reveal magnetic effects more clearly.     

Validation of the new trapped environment AE9/AP9/SPM at low Earth orbit

The completion of the international space station (ISS) in 2011 has provided the space research community an ideal proving ground for future long duration human activities in space. Ionizing radiation measurements in ISS form the ideal tool for the validation of radiation environmental models, nuclear transport codes and nuclear reaction cross sections. Indeed, prior measurements on the space transportation system (STS; shuttle) provided vital information impacting both the environmental models and the nuclear transport code developments by indicating the need for an improved dynamic model of the low Earth orbit (LEO) trapped environment. Additional studies using thermo-luminescent detector (TLD), tissue equivalent proportional counter (TEPC) area monitors, and computer aided design (CAD) model of earlier ISS configurations, confirmed STS observations that, as input, computational dosimetry requires an environmental model with dynamic and directional (anisotropic) behavior, as well as an accurate six degree of freedom (DOF) definition of the vehicle attitude and orientation along the orbit of ISS.     

Preparation of the Biochip experiment on the EXPOSE-R2 mission outside the International Space Station

Biochips might be suited for planetary exploration. Indeed, they present great potential for the search for biomarkers – molecules that are the sign of past or present life in space – thanks to their size (miniaturized devices) and sensitivity. Their detection principle is based on the recognition of a target molecule by affinity receptors fixed on a solid surface. Consequently, one of the main concerns when developing such a system is the behavior of the biological receptors in a space environment. In this paper, we describe the preparation of an experiment planned to be part of the EXPOSE-R2 mission, which will be conducted on the EXPOSE-R facility, outside the International Space Station (ISS), in order to study the resistance of biochip models to space constraints (especially cosmic radiation and thermal cycling). This experiment overcomes the limits of ground tests which do not reproduce exactly the space parameters. Indeed, contrary to ground experiments where constraints are applied individually and in a limited time, the biochip models on the ISS will be exposed to cumulated constraints during several months. Finally, this ISS experiment is a necessary step towards planetary exploration as it will help assessing whether a biochip can be used for future exploration missions.     

The influence of space flight factors on viability and mutability of plants.

The experiments with air-dried Crepis capillaris seeds aboard the Soyuz 16 spaceship and the orbital stations Salyut 5, 6, 7 have revealed an increase in the frequency of aberrant cells in seedlings grown from flight-exposed seeds during the flight (experiment) and after the flight on Earth (flight control) as compared to the ground-based control. The increase in seedlings grown during the flight is more significant than in the flight control. During the flight Arabidopsis thaliana developed from cotyledons to the flowering stage. Analysis of seeds setting on these plants after the flight has shown a reduction in the fertility of these plants and an increase in the frequency of recessive mutants ("Light block-1"). An increased frequency of mutants was also retained in the progeny of plants which had passed through a complete cycle of development during the flight ("Fiton-3"). Suppression of embryo viability was observed in all experiments and expressed itself in reduced germinating ability of seeds from the exposed plants and in the early death of seedlings. Damages resulting from chromosome aberrations are eliminated in the first postflight generation and damages resulting from gene mutations and micro-aberrations are preserved for a longer time.     

Space Shuttle drops down the SAA doses on ISS

Long-term analysis of data from two radiation detection instruments on the International Space Station (ISS) shows that the docking of the Space Shuttle drops down the measured dose rates in the region of the South Atlantic Anomaly (SAA) by a factor of 1.5–3. Measurements either by the R3DE detector, which is outside the ISS at the EuTEF facility on the Columbus module behind a shielding of less than 0.45 g cm⁻², and by the three detectors of the Liulin-5 particle telescope, which is inside the Russian PEARS module in the spherical tissue equivalent phantom behind much heavier shielding demonstrate that effect. Simultaneously the estimated averaged incident energies of the incoming protons rise up from about 30 to 45 MeV. The effect is explained by the additional shielding against the SAA 30–150 MeV protons, provided by the 78 tons Shuttle to the instruments inside and outside of the ISS. An additional reason is the ISS attitude change (performed for the Shuttle docking) leading to decreasing of dose rates in two of Liulin-5 detectors because of the East–West proton fluxes asymmetry in SAA. The Galactic Cosmic Rays dose rates are practically not affected.     

Pion and electromagnetic contribution to dose: Comparisons of HZETRN to Monte Carlo results and ISS data

Recent work has indicated that pion production and the associated electromagnetic (EM) cascade may be an important contribution to the total astronaut exposure in space. Recent extensions to the deterministic space radiation transport code, HZETRN, allow the production and transport of pions, muons, electrons, positrons, and photons. In this paper, the extended code is compared to the Monte Carlo codes, Geant4, PHITS, and FLUKA, in slab geometries exposed to galactic cosmic ray (GCR) boundary conditions. While improvements in the HZETRN transport formalism for the new particles are needed, it is shown that reasonable agreement on dose is found at larger shielding thicknesses commonly found on the International Space Station (ISS). Finally, the extended code is compared to ISS data on a minute-by-minute basis over a seven day period in 2001. The impact of pion/EM production on exposure estimates and validation results is clearly shown. The Badhwar–O’Neill (BO) 2004 and 2010 models are used to generate the GCR boundary condition at each time-step allowing the impact of environmental model improvements on validation results to be quantified as well. It is found that the updated BO2010 model noticeably reduces overall exposure estimates from the BO2004 model, and the additional production mechanisms in HZETRN provide some compensation. It is shown that the overestimates provided by the BO2004 GCR model in previous validation studies led to deflated uncertainty estimates for environmental, physics, and transport models, and allowed an important physical interaction (π/EM) to be overlooked in model development. Despite the additional π/EM production mechanisms in HZETRN, a systematic under-prediction of total dose is observed in comparison to Monte Carlo results and measured data.