Anal. Chem. 1981, 53, 1249-1253 (13) Cotton, F. A. “Chemical Applications of Group Theory”; Wiley-Interscience: New York, 1963; pp 102-104. (14) Becker, R. S.; Singh, I.S.; Jackson, E. A. J . Chem. Phys. 1963, 38, 2144-21 7 I . (15) iaffd, H . H.; Orchin, M. “Theory and Applications of Ultraviolet SDectroscoDv”: Wiiev: New York. 1962: DD 294-344. (16) Murreil, J. ‘N. ‘“The-Theory of the Elect;onlc Spectra of Organic Molecules”; Why: New York, 1963; pp 91-132. (17) Gailivan, J. 8.; Brlnen, J. S. I n “Molecular Luminescence”; Lim, E. L., Ed.; W. A. Benjamin: New York, 1969; pp 93-110. (18) Azumi, T.; McGlynn, S. P. J. Chem. Phys. 1962, 37, 2413-2420. (19) Ham, N. S.;Ruedenberg, K. J . Chem. Phys. 1956, 25, 13-26.
1249
(20) Ham, N. S.; Ruedenberg, K. J . Chem. Phys. 1956, 25, 1-13. (21) Platt, J. R. J . Chem. Phys. 1949, 77, 484-495. (22) Stroupe, R. C.; Tokousbaiides, P.; Dickinson, R. B., Jr.; Wehry, E. L.; Mamantov. G. Anal. Chem. 1977. 49. 701-705.
RECEIVED for review January 5,1981. Accepted April 15,1981. This research was supported in part by National Science Foundation Research Grants CHE77-12542and cHE80-25282 to the University of Tennessee.
Determination of Trace Element Profiles and Concentrations in Human Hair by Proton-Induced X-ray Emission Spectrometry John L. Campbell,” Shatha Falq, Rosalind S. Gibson,’ Sean B. Russell, and Christian W. Schulte Department of Physics, University of Guelph, Guelph, Ontario, Canada N 1G 2 W I
The effect of proton bombardment on the integrity of human hairs is studied via Rutherford scattered protons and emltted characteristic X-rays. At the current densities necessary for rapid PIXE analysis, damage can be minimized by use of a helium atmosphere. The scanning of individual hairs with about 1 mm resbiution and the subsequent conversion of one hair to a thin target are described. The first measurement provides a position dependence of trace element concentrations in relative terms; the second provides absolute concentrations for the entire hair. The position-dependent data can then be converted into concentrations. This provides a history of trace element deposition into the growing hair over a period of several moriths with a time resolution of a few days. An application to druginduced zinc deficiency in human subjects is described.
A human hair consists of three main components as depicted schematically in Figure l. The hard outer cuticle is a layer of scales encasing and protecting the cortex, which in turn is a stable fibrous protein (keratin). In the center of the cortex is a continuous or discontinuous medulla, composed of large cornified cells, loosely linked together; in fine hairs this is often absent. Trace elements are incorporated into the proximal end of a growing hair BS it emerges (at 0.2-0.5 mm per day) from the follicle. Hopps (1) lists the endogenous sources of trace elements in scalp hairs as: (i) the matrix and connective tissue papilla with its blood and lymph vessels (major); (ii) the sebaceous glands in the skin which provide trace elements from body tissues (minor); (iii) the eccrine sweat glands which also provide trace elements from body tissues (minor); (iv) the epidermis (minor). One therefore expects the changes in trace element content along the hair shaft to reflect to some extent the varying trace metal status of the subject; this makes hair a unique biopsy material. Trace element analysis of hair has long been a contentious topic, and many attempts to correlate the trace element content of bulk hair samples with the content of other specific tissues have yielded disappointing results. Part of the problem undoubtedly lies in hair’s susceptibility to exogenous contamination from the atmospheric aerosol and from cosmetics; there are also large variations with age, sex, geography, diet 0003-2700/81/0353-1249$01.25/0
etc. Valkovii: (2) expresses the opinion that poor sampling, improper preparation, and inappropriate statistical analysis are easily recognized in the majority of papers on hair analysis. Nonetheless Hopps (1)finds strong support from a large body of literature for the view that the trace element content of hair does reflect the overall body intake and hence the trace element status. For example, specific well-controlled studies have recently demonstrated strong relationships between changes in trace elements in hair, serum, and the diet during early infancy (3, 4). Most analyses of hair have used either neutron activation or atomic absorption, both destructive techniques generally requiring a minimum of several strands of hair. It is possible with NAA to cut one hair in segments and analyze these to obtain a trace element profile along the hair, but as the segment size approaches a few days’ growth (1-2 mm) sensitivity is lost. The newer technique of proton-induced X-ray emission (PIXE) is nondestructive, and since proton beams can be focussed to 10 pm diameter, it permits longitudinal scanning with a (possible) effective time resolution (in terms of hair growth) of a few hours. Given the mixed record of bulk hair analysis to date, one must ask if elemental profiles along an individual’s hair may be more rewarding in terms of biomedical information than comparison of bulk concentrations among individuals and controls. Two brief early papers demonstrated the potential of PIXE for such profiling. Valkovii: et al. (5) cut single hairs into 14 segments each of 3 cm length and bombarded these with 3-MeV protons; the resulting X-ray spectra showed that elemental X-ray intensities varied markedly along the hairs. Horowitz and Grodzins (6)used a proton microbeam of 25 hm diameter to scan along the hair of an individual who had ingested methylmercury; the mercury profile clearly reflected this event. Workers subsequently applying PIXE to hair analysis have tended to focus on environmental pollution. For example Rendic et al. (7) demonstrated the absorption by hair of the air pollutants lead, bromine, arsenic, and strontium by measuring the X-ray intensities of these elements relative to that of zinc; a monotonic increase in the ratios with distance from the scalp was observed. The nuclear microprobe group (8) at UKAEA’s Harwell Laboratory has performed a large number of hair analyses; one example (9) was concerned with possible ingestion of arsenic after an accident. Henley et al. 0 1981 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 53, NO. 8, JULY 1981
M e d u l l a e Keratinous Cortex Cu ticle
Table I. Typical Elemental Concentrations in Human Scalp Hair ( 2 1 2 ) major elements
46.1 13.6 28.24 7.53 4.14 0.3
C N 0 H
Cuticular Scales On Outer Surface
s
Ca
-0.1 mmFigure 1. Schematic diagram of a human hair.
(10) argued that the hair root was a better indicator of the internal milieu than the shaft (e.g., less prone to contamination) and demonstrated the feasibility of root analysis by PIXE. Less attention has been paid to the other potential application of PIXE scanning, which is to measure the profile of the biologically essential elements such as iron, copper, and zinc in the hope of studying the time dependence of trace element deficiencies. In this context one is often interested not only in the relative intensities of X-rays as a function of position along the hair but also in the absolute concentrations of the elements. If the positional information can be converted to part-per-million concentrations, then that individual’s absolute status may be compared with others. For example an observed progressive decrease in zinc cannot be identified unambiguously as a worsening Zn deficiency unless the data can be expressed as concentrations that can be compared with healthy controls. The problem of converting intensities of proton-induced X-rays to absolute elemental concentrations has recently been addressed by Whitehead (II), who found calculational methods prone to large errors and experimental methods ineffectual. We agree with most of his conclusions. A typical hair is not “thin” relative to proton range; hair thicknesses (diametric) vary from about 6 to 12 mg/cm2, and the range of a 2.5-MeV proton is about 12 mg/cm2 in carbon. The rapidly decreasing proton energy E results in a decreasing X-ray production cross section a(E) along the path. Given a hair of circular cross section, known diameter, and known concentrations of the major elements C, H, N, and 0 (see Table I) and wuming a uniform distribution of trace elements over cross sectional area, one can calculate the X-ray yield by first integrating along one proton path and then over all paths (chords). This gives the yield of X-rays of element i as
where the entrant and exit energies are El and Ez,S(E)is the stopping power, lz is a constant, and ni is the number of atoms
% mass
minor elements
parts per million
Ti
3-24 2.7-1 2.0 8-17 180-250
Fe
cu Zn
of element i; the exponential term caters for X-ray attenuation en route from the interaction point along distance x in the hair toward the detector. In principle then one can compute “corrections” and derive q from Yi. In practice major element concentrations vary somewhat and cross sections of hairs are not circular; moreover Cookson and Pilling (13) have elegantly demonstrated a tendency for trace element concentrations to peak in the outer cortex and to decrease in the central core region. All these factors result in a 90° detector seeing X-rays that are predominantly from the frontal region and whose intensities can be converted to concentrations only with very large uncertainties. In this paper we describe a new, entirely experimental, method for solving this problem and explore various experimental aspects of PIXE analysis of human hair. One particular concern is the integrity of the hair specimen during irradiation by the proton beam. The energy deposited by the beam raises the temperature; material (e.g., parts of the medulla) may be vaporized and trace elements of interest lost; in addition there may be diffusion and rearrangement of trace elements. EXPERIMENTAL SECTION Apparatus. The complete PIXE arrangement, described partially elsewhere (14)is shown in Figure 2. A proton beam
is made uniform by defocusing it and subsequently picking off a central portion with a variable tantalum aperture (Cl,C2). A 10-cm lead wall eliminates the large y-ray background generated by nuclear reactions at the aperture while subsequent graphite collimators remove protons scattered by its edges; a beam of precisely defined shape and uniform intensity in the X Y plane is then incident upon the target. Target chamber A contains a 36-position wheel for specimens deposited on polycarbonate foils which in turn are supported by aluminum frames. The specimen position is viewed by an 80 mm2 X 5 mm Kevex Si(Li) X-ray detector placed at 1 3 5 O to the beam direction; this angle affords a 50% reduction in electron bremsstrahlung background from targets relative to the more widely used 90’ geometry. Targets prepared by digesting hair in acid and pipetting 5-pL droplets onto foils are analyzed in chamber A. However, when a single hair is being scanned by a beam of dimensions 1 mm x 1mm or less, the absolute masses of trace elements within the beam profile are so small that the X-ray detection efficiency must be maximized at any cost. This is done
10 c m
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Flgure 2. Proton beam arrangement for PIXE. The diagram is to scale, with different reduction factors in the Z and radial directions.
ANALYTICAL CHEMISTRY, VOL. 53, NO. 8, JULY 1981
1251
t
I
I
1
I
2
4
6
8
LINEAR
I 10
b
D E N S I T Y hgrmrn)
Flgure 3. Relative fraction R of 2.5-MeV protons scattered from hair In vacuo at 109 OC, as a function of mass per unit length. Current density was below the damage threshold. & o
in chamber B by moving the Si(Li) detector to a 90" angle. Single hairs of any length up to 9 cm are held vertically in a Teflon frame scanned vertically by a vernier device. In either chamber the Si(Li) detector is surrounded by a 25 mm thick tungsten annulus to reduce nuclear y-ray background and views the hair through a 0.4-mm Mylar absorber and an aluminum X-ray collimator to minimize electron bremsstrahlung and characteristic X-ray background. In B, a silicon surface barrier detector is placed at 109O to monitor scattered protons. In either case transmitted proton charge is integrated on a Faraday cup. A HeNe laser beam (wavelength 632.8 nm) can be directed across chamber B to provide a diffraction pattern at each point along the hair. The fringe spacing, determined at 1m distance with a photodiode on a vernier travel, provides a digital determination of the thickness. By scanning a hair strand through the beam, one can check nondestructively for nonuniformities that might give rise to apparently anomalous points on the PIXE scan. Specimen Preparation. Prior to any measurement, each hair specimen was placed in an acid-washed polyethylene tube and washed with 15 mL of a 1% solution of nonionic detergent (Actinox, Sherwood Medical Industries, St. Louis, MO). The detergent solution contains negligible amounts of the analyzed elements, and its use in the washing procedure is not believed to contribute significantly to the trace metal levels in the hair (15,16).The tube was agitated in an ultrasonic water bath for 30 min at room temperature and centrifuged at 1500 rpm for 15 min and the liquid decanted and discarded. The hair was then rinsed three times in distilled, deionized water. After the hair was dried overnight in an oven at 37 "C and cooled in a desiccator, it was weighed with a magnetic balance (estimated error *1 pg in a typical mass of a few hundred micrograms). After weighing, it was again rinsed and dried prior to mounting in target chamber B. Specimen Mass Measurement. Two experiments were undertaken to explore means of measuring variations in the linear density of hair strands. In the first, the ratio R of scattered to transmitted proton intensity was measured for 14 short hair segments whose masses were determined precisely. The results are given in Figure 3. In the second, the relation between diameter and mass of short segments was determined via the fringe spacin, in the diffraction patterns; the results are given in Figure 4. Specimen Integrity. The following experiments arose from the fear that beam heating might partially destroy the hair and volatilize trace elements. First R was measured as a function of increasing current density for one specific hair strand. As proton current from the accelerator was increased, the focusing properties of the beam altered slightly, and so each R value was normalized to that of a nondegradable target, viz., a tungsten wire of 0.05 mm diameter; the results are given in Figure 5. In Figure 6 is a corresponding plot of elemental X-ray intensities as a function of current density. The data of Figures 5 and 6 indicate that the hair is altered as current density increases. The experiments were therefore repeated with the specimens protected by a helium atmosphere (pressure lo4 Pa) which provided a path for heat and charge removal. The helium in chamber B was isolated from the
10
L I N E A R DENSITY
(us,mm)
Flgure 4. The square of hair diameter D (measured from the diffraction pattern of 643.Enm iight) vs. mass per unit length. The diameter scale is in relative units.
.28
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HAIR 1 . 8.4pg)mm
,
1
I
I
6
2
10 CURRENT DENSITY (nA/mm2)
1
I
14
18
c
Flgure 5. Relative fraction R of 2.5-MeV protons scattered from hair in vacuo as a function of proton current density.
t
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0
8
12
CURRENT DENSITY (nAlmmZ)
16
-
Figure 6. Characteristic X-ray intensities from hair as a function of current density using 2.5-MeV protons in vacuo.
beam-line vacuum by a Kapton window of thickness 0.012 mm placed 11cm before the target. Since the beam was diffused by the windown, a 7 cm long carbon collimator was employed to redefine it.
ANALYTICAL CHEMISTRY, VOL. 53, NO. 8, JULY 1981
1252
I 1000;
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Figure 7. Characteristic X-ray intensities from hair as a function of
1 60
35
DISTANCE FROM DISTAL E N D (mm)
Figure 9. Zn X-ray intensity as a function of distance from the distal end of a hair from a healthy individual. Error bars are f3a.
current density using 2.5-MeV protons in helium. Each datum was taken at 1 nA/mm2 in vacuo following a 2-min irradiation in helium.
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DISTANCE FROM DISTAL E N D (mm)
Figure 8. Characteristic X-ray intensity as a function of distance from the distal end of a hair taken from an individual undergoing treatment with ethambutol. The right-hand vertical scale gives thhe elemental concentrations derived as described in the text. The errors bars are 1 standard deviation (from counting statistics).
As the scattered proton measurement can only be done under vacuum, the relationship between scattered fraction R and current density was obtained by performing a backscatter measurement in vacuo at 1 nA/mm2 after successive exposures in helium to currents ranging from 1 to 22 nA/mm2. The results showed no detectable change in R; the Fe, Cu, and Zn K a X-ray intensities per unit proton charge also remained constant over the range of current densities explored, as shown in Figure 7. Longitudinal Scanning. To illustrate the application of longitudinal PME scanning of human hairs, a specimen was taken from a 45 year old female tuberculosis patient 72 days after the commencement of ethambutol treatment. X-ray spectra were recorded for 27 segments at 1.6 mm intervals using 2.5-MeV protons in a lo4Pa helium atmosphere. The beam diameter was 1 mm and the current density 10 nA/mm2. Such a scan takes about 40 min to perform. The position dependence of Fe, Cu, and Zn Kcu X-ray intensities is presented in Figure 8. For comparison, Figure 9 shows a zinc scan from a healthy female subject, the data being taken from points 3.2 mm apart along the shaft. Single Hair Analysis. To convert data such as those of Figure 8 to concentrations per unit mass, we developed the following technique for analysis of a single strand. The aim was to convert the strand to a thin specimen suitable for PIXE analysis without the need for thick-target corrections. After being scanned, the hair strand was transferred to a specially designed Teflon vessel, which had been previously cleaned with nitric acid followed by distilled deionized water. This consisted of a Teflon block in which was drilled a 4 mm diameter hole, 11 mm deep, with a volume of 136 pL. Forty microliters
ANALYTICAL CHEMISTRY, VOL. 53,
Table 11. PIXE Analysis of a Digested Hair (Mass = 400 ~ g Exposed ) to 30 pC/cm2 of 2.5 M e V Protons X-ray intensities blank concn in hair, element specimen (mean of 2) PPm Fe 8 1 4 %50 3 2 3 %100 24.0 % 5.5 Ni 360k 35 3 0 %20 20 ?: 2.5 Cu
Zn Y
2 2 5 %25 1683i: 52 3251 % 58
4 0 %15 1 6 5 %25 3228 % 58
13.5 % 2.5 143 % 5.5
method, data such as those of Figure 8 are converted to concentrations by mass.
RESULTS AND DISCUSSION The data of Figure 3 establish that the ratio R of backscattered proton intensity to transmitted beam provides a measurement, albeit with mediocre sensitivity, of the mass per unit length of hair under irradiation in chamber B. We present a much more detailed study of scattered proton spectra and intensities elsewhere (18). The direct determination of diameter provides a more sensitive determination of linear density, but any given strand may deviate significantly from this “calibration”. Microscopic examination showed that although medullae were absent for most of the specimens of Figure 4,at least two (circled points) were medullated, giving them increased density and thus causing deviation from the calibration. These two methods do not provide a precise and unambiguous mass measurement for a section of a strand undergoing analysis. They do, however, permit a check for variations or anomalies in linear density and are useful adjuncts to the main (X-ray) measurement. Figure 5 shows that during irradiation in vacuo increase in proton current density alters the backscatter response at about 6 nA/mm2. Similarly, Whitehead (11) has reported visual changes in the hair structure above 4.5 nA/mm2. These changes are accompanied here by an increase in the elemental X-ray intensities. One might expect to see a decrease as trace elements are volatilized. However, the observed increase is consistent with a decreased overall specimen mass, resulting in a higher mean proton energy and hence a higher overall X-ray production cross section across the hair. Unfortunately this effect masks any real decrease in trace metal content due to volatilization. (On naive arguments concerning target mass one might have expected R to decrease. One explanation of the observed increase is that the mean atomic number of the remaining material is higher than the value prior to irradiation.) We conclude that X-ray intensities recorded in vacuo should be regarded as suspect. Not only is there significant damage to the hair at currents above 5 nA/mm2but there may be local redistribution of trace elements. Unfortunately the apparently acceptable current levels are not sufficient to provide sufficient precision in counting times that we deemed acceptable. Analysis under vacuum was therefore not pursued further. When the hair was protected by helium both the backscatter fraction and the characteristic X-ray intensities remained constant at current densitities up to 22 nA/mm2. Hence a single hair can be scanned by the proton beam in a helium atmosphere, without loss of integrity, and the profile of elemental X-ray intensities along its length obtained. Taking the typical growth rate as 0.33 mm/day, use of a 1mm X 1mm beam moved in 1mm steps would provide data points 3 days apart in terms of trace element status. The means adopted here to convert the profile data into concentrations are to digest the entire hair in nitric acid after
NO. 8,
JULY 1981
1253
scanning and to prepare from this a thin target for conventional PIXE analysis. The results of Figure 10 and Table I1 demonstrate that it is possible to perform a reliable analysis for several trace metals in a single strand. There is some room for improvement in the trace metal content of our blank, but since our own current interest is in zinc, the blank is for the moment acceptable. The data of Figure 8 for a T B patient undergoing ethambutol (EMB) treatment provide a concluding illustration of the methods developed here. The raw X-ray intensities show a progressive fall in zinc concentration. The overall concentrations of 79 ppm for Zn, 6 ppm for Cu, and 5.1 ppm for Fe provided a recalibration of the vertical scale of Figure 8 in terms of concentration. Neutron activation analysis performed prior to EMB treatment gave initial concentrations of 160 ppm Zn and 10.8 ppm Cu. Our data clearly illustrate a progressive decline in zinc status. This trend was not unexpected, Delacoux et al. (19) having reported a correlation between plasma Zn levels and ocular damage in patients treated with EMB.
CONCLUSIONS The combination of PIXE scanning and subsequent PIXE analysis of single hair strands provides a powerful and painless tool for monitoring trace element status. Some more preparatory work remains necessary. The extent to which any one hair represents the bulk is not known; we therefore plan detailed intercomparisons of a large number of hairs from the scalp of a single subject. ACKNOWLEDGMENT We are deeply grateful to W. E. Morton, T. Riddolls, and G. H. Willis for their contributions to design and construction of equipment. Technical assistance was also rendered by M. Berry and J. Marsh. Discussions with R. W. Ollerhead, I. L. Gibson, and J. R. MacDonald were of great value. We thank R. H. Stinson for suggesting the laser measurement of hair diameter, N. F. Mangelson for providing the poly(viny1 pyrrolidone) treatment for polycarbonate foils, and L. Kahama for providing the hair sample from the T B patient. LITERATURE CITED Hopps, H. C. Sci. Total Environ. 1977, 7 , 71-89. ValkoviE, V. “Trace Elements in Human Hair”; Garland STPM Press: New York and London, 1977. Gibson, R. S.; DeWolfe, M. S. Nub. Rep. Int. 1980, 21, 341-349. Gibson, R. S.; DeWolfe, M. S. Acfa Paediatr. Scand., In press. ValkoviE, V.; Miljanic, D.; Wheeler, R. M.;Liebert, R. B.; Zabel, T. H.; Phillips, G. C. Nature (London) 1973, 243, 543-544. Horowitz, P.; Grodzins, L. Sciene 1975, 189, 795-797. Rendic, D.; Holjevic, S.; ValkoviE, V.; Zabel, T. H.; Phillips, G. C. J . Invest. Dermatol. 1976, 66, 371-375. Cookson, J . A.; Ferguson, A. T. G.; Pllllng, F. D. J. Radioanal. Chem. 1972, 12, 39-52. Houtman, J. P. W.; de Bruin, M.; de Goeij, J. J. M.;Tjioe, P. S. Nuclear Activation Techniques in the Life Sciences”; IAEA: Vienna, 1979; p 599. Henley, E. C.; Kassouny, M. E.; Nelson, J. W. Science 1977, 797, 277-278. Whitehead, N. E. Nucl. Instrum. Methods 1979, 164, 381-388. Report of the Task Group on Reference Man (ICRP 23); Pergamon: Oxford, 1975. Cookson, J. A.; Pllling, F. D. Phys. Med. Biol. 1975, 20, 1015-1020. Campbell, J . L.; Russell, S. B.; Faiq, S.; Schulte, C. W.; Ollerhead. R. W.; Gingerich, R. R. Presented at the Proceedings of the Second International Conference on Partlcle-Induced X-ray Emission and its Analytlc Applications (Lund, Sweden, 1980); Nucl. Insfrum. Methods, in press. Gibson, R. S.; DeWolfe, M. S. Pediatr. Res. 1979, 13, 959-962. Shapcott, D. Clin. Cbem. 1978, 24, 391-392. Mangelson, N. F., private communication. Campbell, J. L.; Faiq, S.; Gibson, R. S.; Russell, S. B. Nucl. Insfrum. Methods 1980, 178, 601-606. Deiacoux, E.; Moreau, Y.; Godefroy, A,; Evstigneef, T. J. h. Opthalmol. 1978, 1 , 191-196.
RECEIVED for review January 23, 1981. Accepted April 20, 1981. This work was supported by the Natural Sciences and Engineering Research Council of Canada.
