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Preliminary Investigation of Proton and Helium Ion Radiation Effects on Fluorescent Dyes for Use in Astrobiology Applications Daniel P. Thompson,*,† Paul K. Wilson,‡ Mark R. Sims,† David C. Cullen,‡ John M. C. Holt,† David J. Parker,§ and Mike D. Smith§
Space Research Centre, Department of Physics & Astronomy, University of Leicester, University Road, Leicester LE1 7RH, U.K., Cranfield Biotechnology Centre, Institute of BioScience and Technology, Cranfield University, Silsoe, Bedfordshire MK45 4DT, U.K., and School of Physics and Astronomy, University of Birmingham, Edgbaston, Birmingham B15 2TT, U.K.
The Specific Molecular Identification of Life Experiment (SMILE) instrument (Sims et al. Planet. Space Science 2005, 53, 781-791) proposes to use specific molecular receptors for the detection of organic biomarkers on future astrobiology missions (e.g., to Mars). Such receptors will be used in assays with fluorescently labeled assay reagents. A key uncertainty of this approach is whether the fluorescent labels used in the system will survive exposure to levels of solar and galactic particle radiation encountered during a flight to Mars. Therefore, two fluorescent dyes (fluorescein and Alexa Fluor 633) have been exposed to low-energy proton and r radiation with total fluences comparable or exceeding that expected during an unshielded cruise to Mars. The results of these initial experiments are presented, which show that both dyes retain their fluorescent properties after irradiation. No significant alteration in the absorption and emission wavelengths or the quantum yields of the dyes with either radiation exposure was found. These results suggest other structurally similar fluorophores will likely retain their fluorescent properties after exposure to similar levels of proton and r radiation. However, more extensive radiation fluorophore testing is needed before their suitability for astrobiology missions to Mars can be fully confirmed. One of the key aims of planetary research is to understand the organic inventory on other planets and in particular to search within the organic inventory for evidence of extinct or extant life. A number of instruments are under development that will look for specific organic molecules and molecular types within this context by exploiting modern developments in analytical technologies from the biosciences sector. One such instrument concept is the Specific Molecular Identification of Life Experiment (SMILE) family of instruments1 under development for the European Space * Corresponding author. E-mail:
[email protected]. † University of Leicester. ‡ Cranfield University. § University of Birmingham. (1) Sims, M. R.; Cullen, D. C.; Bannister, N. P.; Thompson, D. P.; Wilson, P. K. Planet. Space Sci. 2005, 53, 781-791.
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Agency (ESA) ExoMars astrobiology mission.2 These types of instruments, which have been called “life marker chips”, and their use in the proposed missions are based on the use of specific molecular receptors and associated detection systems to determine the presence of molecular biomarkers within an astrobiology/planetary exploration context. Molecular receptors can be based on biological systems or artificial (i.e. chemistry-based systems). In the short term, the use of antibodies (biologically derived molecular recognition elements) as highly specific receptors for astrobiology target molecules is preferred to synthetic approaches due to their typical higher specificity, higher binding affinities, and proven use in related terrestrial applications. The SMILE instrument will detect specific biomarkers using antibody binding assays with appropriate assay components labeled with fluorescent dyes. During transfer to Mars, a spacecraft will be exposed to a wide range of radiation types and energies including γ rays, protons, neutrons, and heavy ions (e.g., Adams3 and Petrov4) at energies ranging from a few MeV to thousands of GeV and beyond, peaking at around 1 GeV (see, for example, Benton and Benton5). The success of a SMILE-type approach to biomarker detection will rely on the survival of the antibodies and fluorescent dyes during the flight to Mars and operations on the Martian surface. To the authors’ current knowledge, there have not been any reported studies of fluorescent dye stability in such radiation environments. Therefore, it is imperative that representative radiation exposure studies are performed to demonstrate that the key components of biomarker detection (antibodies and fluorescent dyes) can survive such radiation environments before substantial instrument development work is performed. Thus, preliminary studies for two types of fluorescent dyes (fluorescein and Alexa Fluor 633, see Figure 1) are reported. The dyes were exposed to low-energy proton and R beams from a cyclotron with total fluences comparable or exceeding that (2) Vago, J. L.; Gardini, B.; Kminek, G.; et al. ESA’s new mission to search for signs of life on Mars: ExoMars and its Pasteur scientific payload, EGSAGU-EUG Joint Assembly, 2003 [meeting report]. (3) Adams, J. H., Jr. Nucl. Tracks Radiat. Meas. 1992, 20, 397-40. (4) Petrov, V. Adv. Space Res. 2004, 34, 1451-1454. (5) Benton, E. R.; Benton, E. V. Nucl. Instrum. Methods B 2001, 184, 255294. 10.1021/ac052206u CCC: $33.50
© 2006 American Chemical Society Published on Web 02/24/2006
Figure 1. Chemical structures of fluorescein (left) and Alexa Fluor 633 (right).
expected during an unshielded cruise to Mars. The dyes’ fluorescent properties were measured before and after exposure. The studies are performed with dry fluorescent dye samples as this represents the expected format for fluorescent dyes in a final life marker chip instrument with hydration only occurring within an aqueous sample upon final use. Studies with other space radiation components and studies of the radiation effect on antibodies will be reported elsewhere. MATERIALS AND METHODS Radiation Sources. Proton and R particle (helium ion) exposures were carried out at the cyclotron facility at University of Birmingham (Birmingham, UK). The cyclotron (Scanditronix MC40, Scanditronix, Sweden) was capable of generating particles at any specified total kinetic energy between 9 and 35 MeV in vacuo to within approximately ( 50 keV (fwhm) and with variable beam current enabling a large range of fluences to be achieved within an acceptable time period. Sample Preparation. Due to the cross-sectional area of the particle beam (approximately 1 cm2) emerging from the cyclotron beam line exit port, tests had to be conducted using bespoke sample positioning holder and containers. Thus, a simple metal holder and adaptor plate were fabricated to attach to existing spigots on the cyclotron beam line exit port. The holder accepted polystyrene microwell blocks cut from 864 well microplates (Genetix Ltd., New Milton, UK). The blocks comprised 12 mm × 12 mm sections each containing 9 wells and with a maximum well volume of 15 µL (see Figure 2). To avoid contamination, blocks were prepared and cleaned by immersion with ultrasonication in 1% v/v Teepol detergent in deionized water solution for 5 min, followed by immersion and ultrasonication in hot deionized water for 5 min, followed by two cycles of immersion and ultrasonication in fresh deionized water for 5 min, and finally drying with a nitrogen gas stream and baking dry (at 80 °C). Three 10 µL samples of 1.0 mg‚mL-1 fluorescein sodium salt (Sigma Aldrich, Poole, UK) and three 10 µL samples of 0.1 mg‚mL-1 Alexa Flour 633 (Invitrogen Ltd., Paisley, UK) in deionized water were placed in wells in each 9 well block, in a random, known pattern. The sample blocks were then placed into a desiccator vessel containing excess self-indicating silica gel desiccant, and this vessel was placed into an oven at 37 °C for 24 h. This resulted in the evaporation of all the water from the sample blocks, leaving
Figure 2. Photograph taken after radiation testing of six 12 mm × 12 mm sections of an 864 well microplate (containing 9 wells per section) showing the format for the fluorescent dye exposures. The blocks are further located in a holder that interfaces to the exit port of the cyclotron beam line. Block number 3 (image bottom right) has a reflective glassy surface that is assumed to be a result of heating due to a high beam current (500 nA) as compared to a low beam current (50 pA) for the remainding five blocks.
only dried fluorescent dye in the wells. The sample blocks were then individually wrapped in aluminum foil to reduce ambient light induced photo-bleaching and kept at 5 °C in a sealed container with excess self-indicating silica gel desiccant, until transportation to the cyclotron facility in the same sealed box at ambient temperature. Exposure Calculation. The cyclotron facility generates a mono-energetic proton beam rather than the broad energy spectrum, which would be observed in space. To consider possible energy-dependent radiation effects, the energy spectrum was broken into two parts; 9-35 MeV and 35 MeV upward. These boundary energies (9 and 35 MeV) were chosen because they were the theoretical minimum and maximum particle energies for which the cyclotron could create a stable beam. Using the SPace ENVironment Information System (SPENVIS) (European Space Agency General Support Technologies Programssee www.spenvis.oma.be), the total integrated fluence of protons was calculated over all energies to a 95% confidence level for an EarthMars transit (see Supporting Information). The Earth-Mars transit parameters were based on those suggested in the Mars Transportation Environment Definition Document,6 which uses a transit configuration starting January 25, 2014, and finishing July 22, 2014, with therefore a total time of 179 days. The entire transit (6) Alexander, M., Ed. Mars Transportation Environment Definition Document NASA/TM-2001-210935; 2001.
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takes place during the “on” season for solar flares within the Jet Propulsion Laboratory interplanetary proton fluence model7 and therefore represents a likely worst case scenario. The integrated fluence was divided at the boundaries stated and the necessary cyclotron current calculated to achieve exposure to the required fluence in a reasonable time (up to a few hours). This approach gave an estimated mission dose of 1.2 × 108 protons with energies between 9 and 35 MeV and 3.12 × 107 protons with energies higher than 35 MeV. The test samples were exposed to various multiples of these basic fluences, specifically, 1×, 10×, 100×, and 10 000×, for the 9 MeV proton tests and 1× and 100× for the 30 MeV particle tests. For R particle tests, the mission R particle dose was not calculated, although it would be expected to be significantly less than the proton fluence. To further enhance the “over-test” principle adopted within this work, it was assumed that the R particle fluence was the same as the proton fluence with values of 1× and 100× tested. Cyclotron beam currents of 50 pA and 500 nA were used to achieve the required fluences. To validate the exposures and assess the uniformity of the proton/R particle beams across the exposed sample area, radiochromic film was exposed to the same fluence of protons/R particles. Sample Exposure. The cyclotron was set up to produce a particle beam of the desired energy and current. The sample blocks were removed from their foil wraps and placed into a custom-built aluminum holder, which was then mounted in front of the particle beam such that the sample block was directly in the beam line with the uncovered wells facing toward the particle beam. All tests were therefore conducted in an ambient atmosphere at room temperature and pressure. Other tests planned will address the question whether the local environment (presence of molecular oxygen, water vapor, vacuum, etc.) affects the results. The cyclotron chamber was then sealed, and the samples were exposed to the appropriate particle beam for the appropriate period of time to achieve the desired fluence for each test. The beam current, and hence the fluence and exposure, was controlled and measured by inserting a Faraday cup into the beam before and after the sample block exposure. Following exposure, the sample blocks were then removed from the mounting, wrapped in aluminum foil, and stored again in a sealed container with excess self-indicating silica gel desiccant. The samples were then transported to Cranfield University for testing. After each sample exposure, a piece of radiochromic film was exposed to the same fluence of particle to validate the exposure and measure the uniformity of the particle beam to ensure all the samples in each block were exposed to an equal fluence. The results from these films tests (not shown) confirmed that the beam had been uniform across the exposure area and samples for each test conducted. Upon commencement of testing, the cyclotron was unable to achieve a stable 35 MeV beam, so the higher energy test was run at 30 MeV while exposure times were kept the same. The difference between predicted mission fluences allowing for this change in energy range is 0.2 × 109 particles, approximately an 11% increase for a 30 MeV upward energy range as compared to a 35 MeV and upward range. This reduction in energy was thought to make the test more stringent due to the inverse relationship
between particle energy and Linear Energy Transfer (LET) value within this part of the energy spectrum for water, which was taken at the nearest reference material for which data was available. (Note: LET is a measure of the energy deposited per unit length in a given material.) Additionally, it should be noted that all the beam energies quoted were for the beam in vacuo; therefore, the actual energies at the target surface were expected to be reduced as the particle beam passed through a 25 µm Havar window (an alloy of cobalt, chromium, nickel, and iron) followed by approximately 1 cm of air before reaching the samples. For the 30 MeV protons this energy change is negligible (reduced to ∼29.8 MeV8), so their LET remains essentially unchanged at ∼1.9 keV/µm. For the 9 MeV protons, the window and air lead to ∼6% increase in LET from the vacuum value to around 5.4 keV/µm. The R particles are affected the most, their energy dropping to ∼26.9 MeV, with an LET of approximately 26 keV/µm. Due to the increased linear energy transfer values for particles, a stronger interaction between the beam and sample was expected, adding to the over-test philosophy. Sample Analysis. The fluorescent dyes were reconstituted by adding 15 µL of deionized water to each well of the sample block and allowing the dyes to dissolve for 1 min. The solution was then removed from the sample block and added to a vial of 925 µL of deionized water. Each well was then washed by four further cycles of 15 µL deionized water, allowing time for dissolution of remaining dye, and adding the solution to the vial. Therefore for each different exposure this produced three vials containing 1 mL of 0.1 µg‚mL-1 fluorescein and 1 mL of 0.1 µg‚mL-1 Alexa Fluor 633 solutions. These samples were analyzed in a fluorescence spectrophotometer (Cary Eclipse Varian controlled with Cary Eclipse Scan software, version 1.0(78), (Varian Inc., Palo Alto, CA)), with each sample being processed three times under identical conditions to reduce errors. Data were taken with the instrument software in its default settings, with the following exceptions: the scan speed was set to slow (120 nm‚min-1); the emission and excitation slit widths were set to 5 nm; and the detector voltage (photomultiplier tube gain) was set manually to an appropriate level for each dye (450 V for fluorescein and 500 V for Alexa Fluor 633).
(7) Feynman, J.; Armstrong, T. P.; Dao-Gibner, L.; Silverman, S. M. J. Spacecraft Rockets 1990, 27, 403-410.
(8) Parker, D. University of Birmingham, UK, Private communication, January 31, 2006.
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RESULTS To assess any degradation of the fluorescent dyes after exposure to the various particle radiations, the optical properties of the dyes were assessed for changes in peak wavelength and changes in the intensity of fluorescence emission. Changes in peak wavelength of emission would indicate subtle changes in the structure of the dyes that still retain fluorescence properties, while changes in the intensity would indicate more gross degradation of the dyes. Figure 3 shows the peak fluorescence emission wavelength of fluorescein after each of the radiation exposures. It is apparent that the peak fluorescence emission wavelength of fluorescein does not appear to be significantly affected by 10×, 100×, and 10 000× the calculated mission fluence of protons at the stated energies for the chosen Mars flight. In addition, exposure to R
Figure 3. (Left) Peak emission wavelength of fluorescein fluorescence at an excitation wavelength of 488 nm after exposure to proton and R particle radiation. Error bars show the standard error from the mean values (n ) 3). Estimated mission dose ) 1.2 × 108 protons for 9 MeV exposures and 3.12 × 107 particles for 30 MeV exposures. (Right) Peak intensity of fluorescein fluorescence emission using an excitation wavelength of 488 nm after exposure to proton and R particle radiation exposure. Error bars show the standard error from the mean values (n ) 3).
Figure 4. (Left) Peak emission wavelength of Alexa Fluor 633 fluorescence at an excitation wavelength of 633 nm after exposure to proton and R particle radiation exposure. Error bars show the standard error from the mean values (n ) 3). (Right) Peak intensity of Alexa Fluor 633 fluorescence emission at an excitation wavelength of 633 nm after exposure to proton and R particle radiation exposure. Error bars show the standard error from the mean values (n ) 3).
particles at 1× or 100× the expected proton fluence (a significant over estimation of the number of R particles that would be encountered during the mission) does not appear to have affected the peak fluorescence emission wavelength either. Similarly, the fluorescence emission intensity as shown in Figure 3 does not show any significant change in value with varying radiation fluence or type. Note that the 1× fluence data for proton exposure is not present due to an oversight during sample preparation. Similarly, the peak absorption wavelength and absorption efficiency of fluorescein were also found to be unaffected by these radiation exposures (data not shown). Figure 4 shows the peak fluorescence emission wavelength of Alexa Fluor 633 after each of the radiation exposures. It is also apparent that the peak fluorescence emission wavelength of Alexa Fluor 633 does not appear to be significantly affected by 10×, 100×, and 10 000× the calculated mission fluence of protons at the stated energies for the chosen Mars flight. In addition, exposure to alpha particles at 1× or 100× the expected proton fluence does not appear to have affected the peak fluorescence emission wavelength either. The fluorescence emission intensity as shown in Figure 4 does not show any significant change in
value but does show evidence of a slight increase in emission intensity at 10× and a decrease at 10 000× mission fluence. Note that again the 1× fluence data for proton exposure is not present due to an oversight during sample preparation. There also appears to be a slight enhancement of fluorescence emission upon increasing R particle fluence. Similarly, the peak absorption wavelength and absorption efficiency of Alexa Fluor 633 were also found not to show any significant changes in fluorescent properties at the various radiation exposures (data not shown). It was also noted that the sample block for the 10 000× proton exposure (23 s at 500 nA) displayed a top surface that took on a reflective “glassy-like” property as shown in Figure 2. It is assumed this is due to heating and resultant partial melting of the polymer during the exposure; therefore, samples in this block may have been exposed to temperatures in excess of 140 °C for a few seconds in addition to the proton exposure. DISCUSSION The testing described in this work was based on a number of necessary assumptions and simplifications. It was not possible to simultaneously replicate all aspects of the interplanetary environAnalytical Chemistry, Vol. 78, No. 8, April 15, 2006
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ment inside the laboratory. Therefore, individual aspects needed to be investigated in isolation, starting with radiation and initially via testing with mono-energetic beams. It is additionally important to understand the sensitivity of fluorescent dye and also antibody components to the various radiation types in isolation in order to design appropriate levels and types of radiation shielding for a final instrument and spacecraft if required. In a deep space mission environment there are two major particle radiation components: solar particle events and galactic cosmic rays (GCR). In terms of particle numbers, by far the most common type of particle radiation in the interplanetary environment is solar proton radiation. The GCR are generally much higher energy than the radiation used in these tests. R particle tests were conducted as a first step in simulating damage due to ions more commonly present in GCR. None of the tests included the rate of exposure (cyclotron current) as a factor, and it was assumed this would not have an effect upon the results. It is obviously unfeasible to conduct the tests at low rate over the same time scale (≈6 months) as the actual mission, and so some inconsistency between reality and the test was inevitable. It was also not possible to conduct all tests at the same current as the total fluence needed to be and was varied by many orders of magnitude. These higher fluences would have involved testing over several days if the same beam current chosen for the lower fluence tests was used, which was again unfeasible. It should be noted that all space component radiation tests are normally conducted as these tests were and make the same assumption that any effects are rate independent. It is intended that future work will include rate effect testing to validate this assumption. Additionally to test any synergistic effects of radiation in a real space environment, it is planned that SMILEtype instrument reagents can will fly on ESA’s Biopan space exposure facility (see, for example, Horneck9) in 2007, where among other things the exposure rate will be more realistic. This mission will however be in the near Earth environment rather than a deep space radiation environment. The exposure fluences were simplified from the actual energyfluence spectrum calculated to accommodate the fact that (a) the cyclotron could only operate at one energy at any one time and (b) the cyclotron could only theoretically operate between 9 and 35 MeV (due to operational difficulties it was only able to reach 30 MeV, but this was not known at the time of calculating the exposures). This given, it was decided to make maximum use of the energy range available and use the highest and lowest energy values for the tests. The calculated fluence was then simply divided at these two energies. The two obvious disadvantages to this are that it does not incorporate the effect of (a) the largest part of the solar proton fluence (i.e., that with an energy of less than 9 MeV) and (b) the highest energy protons with an energy of greater than 35 MeV. However, it should be noted that while GCR and solar protons are generally much higher energy than the radiation used in these tests, the actual life marker chip device would be positioned inside a spacecraft, where lower energy secondary radiation and fragments from the primary radiation will play a large role. Also, the spacecraft shielding is likely to prevent much damage from the lowest energy primary particles, making the energy range the tests were conducted in far more applicable (9) Horneck, G. Planet. Space Sci. 1999, 23, 381-386.
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than might initially be assumed. To remove the effect of shielding, which can vary considerably with spacecraft and mission design, and to better understand the effects of radiation, unshielded fluorescent dyes and antibodies were irradiated. It was decided that from a mission scenario point of view a “worst case” test plan should be adopted. This meant that rather than using the average energy by integrated fluence over the defined ranges (9-35 MeV and >35 MeV), the lowest energy of that range was used (see Supporting Information, Figure A). Noting that the LET value for protons in water peaks at low energies ∼0.1 MeV (see, for example, Date et al.10), it was assumed that using the lowest energy in the range would be potentially more damaging to the sample than if it were exposed to the same fluence using the range of (higher) energies. Given the range of protons in water these beam energies were sufficient to deposit energy throughout the samples. Consequently, despite the limitations, it is believed that the results indicate survival after “worst case” radiation exposures. Studies have shown (for example, Lee et al.11 and Cho and Song12) that proteins without the presence of water or another liquid are less susceptible to radiation damage. It is believed hydroxyl radicals, created by the interaction of the radiation with a water medium, are the major cause of radiation damage within the protein (for example, Yamaguchi et al.13). Without the solvent material and therefore the means to create these radicals, the major damage mechanism is removed. The results obtained by these tests, which show no damage under high radiation doses, appear to vindicate the baseline instrument design for SMILElike life marker chips of transporting the biological assay reagents in desiccated form and are consistent with other work.11,12. There were a number of other obvious limitations inherent in the tests. First, they were not performed in a vacuum, under microgravity, or at the extremely cold temperatures as would be the case in a spaceflight scenario. The assumption was made that the ambient environment in the test chamber (normal atmospheric pressure and ambient humidity, Earth gravity, and room temperature) would either not effect the results obtained or be detrimental to the samples, as water vapor and oxygen would be present, perhaps inducing damage under radiation (see above). Further testing under space environment conditions is being planned and pursued. It is also noted that leaving the wells uncovered and exposed to the ambient environment could allow sputtering of the samples and that ionization of the air could cause further transport between samples. Future testing will likely include a thin film cover for the samples to prevent such events from occurring and will include analysis of chemical changes within the target materials. Another factor not yet taken into account is how the chemical link between fluorophores and the antibodies/analytes will be affected by radiation. The ability of the fluorophore to remain linked to an antibody/analyte is just as important as its ability to retain fluorescence after exposure to radiation, and so further investigation of this will also be conducted. (10) Date, H.; Sutherland, K. L.; Hayashi, T.; Matsuzaki, Y.; Kiyanagi, Y. Radiat. Phys. Chem. 2006, 75, 179-187. (11) Lee, S.; Lee, S.; Song, K. B. Food Chem. 2003, 82, 521-526. (12) Cho, Y.; Song, K. B. J. Biochem. Mol. Biol. 2000, 33, 133-137. (13) Yamaguchi, H.; Uchihori, Y.; Yasuda, N.; Takada, M.; Kitamura, H. J. Radiat. Res. 2005, 46, 333-341.
Despite these limitations, it is believed that the tests represent a valid worst case initial radiation test of one of the critical components of a life marker chip instrument. Future testing will include heavy ions representative of GCR (Fe, Ti, Si, C, O, etc.), neutron exposure, and electromagnetic radiation (γ and X-ray). Some initial γ ray testing has been conducted, and the results will be reported separately. SUMMARY A major requirement for the potential application of biological assays in astrobiology has been demonstrated in this research. An experimental procedure for the exposure of fluorescent materials (and also for biological materials such as antibodies) to proton and R particle radiation has been developed. This procedure allows multiple samples to be exposed to each radiation treatment and therefore allows assessment of the reproducibility of both the sample preparation and extraction procedure and any effect of the radiation on the samples. It has been found that two fluorescent dyes suitable for applications in biological assays have not been significantly affected by proton and R particle exposures up to several orders of magnitude greater than they would encounter during the suggested 6-month space flight to Mars. 6 months was chosen as a typical “high-speed” Earth-Mars transfer trajectory, applicable to the majority of Mars missions. One of the first possible opportunities to fly a life marker chip is the ESA ExoMars mission. While ExoMars plans involve a 23-24 month cruise phase, the
over-testing of the samples by several orders of magnitude means the results quoted here are applicable to such increased duration missions. It can therefore be hypothesised that other structurally similar fluorescent dyes are also unlikely to be damaged by these radiation conditions. These results are a step toward validating the utilization of highly specific antibody-based assays (i.e., life marker chips) in astrobiology applications as proposed for the SMILE instrument. ACKNOWLEDGMENT D.P.T. acknowledges the departmental studentship provided by the University of Leicester Space Research Centre. P.K.W. was supported via an EPSRC studentship. This research was partially supported by funds provided by Dr. M. R. Sims of the University of Leicester. The authors thank the referees for their helpful suggestions and inputs to this paper. SUPPORTING INFORMATION AVAILABLE SPENVIS orbit generation and Solar Proton Event figures. This material is available free of charge via the Internet at http:// pubs.acs.org.
Received for review December 14, 2005. Accepted February 6, 2006. AC052206U
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