Nuclear microprobe analysis of 14N and 15N in soybean leaves

Nuclear Instruments and Methods in Physics Research B 82 (1993) 465-473 North-Holland

Nuclear microprobe

NDMB

Beam Interactions with Materials&Atoms

analysis of 14N and ““N in soybean leaves

P. Massiot ‘, V. ~ic~aud ‘, F. Sommer b, N. Grignon ‘, A. Gojon ‘, C. Ripoll d and M. Thellier d a Laboratoire “Piem Siie” CEA /DSM/DRECAM CE Saclay, 91191 Gif-sur-Yvette cedtx, France b Science et Surjace, CENATS, Unirrersith Claude Bernard, 69622 ViNeurhanne, France ’ Laboratoire de Biochimie et Physiologic V&t!tales INRA-CNRS URA 573, Ecole Nationale Sup&twre Agronomique, 34060 Mon tpelkr, France ” ~rato~re des Procesws ioniqtm ~~U~~~~es CNRS WA 203, Fact&e’ des Sciences de Rouen, 76134 M’ S’ A&m

cedex, France

Received 29 January 1993 and in revised form 25 March 1993

A combination of hvo nuclear microprobe exploitation modes of spectroscopy has been developed to detect ‘“N and “N in ray emission plant tissues. l”lN was determined by proton elastic scattering analysis (PESA) and “N by proton induced bails (PIGE). The advantages of each mode are outlined. The preparation meth~ of the soybean samples which allows the preservation of the tissue structure is described. After checking the linearity of the quantitative evaluations using calibration curves, we have studied the irradiation damages, evahlating the nitrogen mass loss with the nuclear microprobe facilities of Bruyires-le-Chstcl. An experimental approach for quantitative 14N and “N analysis by these nuclear methods in ctyofiied and resin embedded soybean leaves is proposed.

1. Introduction

Plants absorb mineral forms of nitrogen, mainly nitrate, and they reduce them to organic nitrogen. This nitrogen metabolism of plants, which is among one of the most essential processes in the general c~onom~ of life, involves the cooperation of various tissues, cells and subcellular organelles. As stressed by metabolic and transport studies [l], it is therefore important to label the different chemical forms of nitrogen and to detect, image and quantify nitrogen and its isotopes in the plant tissues with the best possible ~~si~i~ty and resolution. There arc several physicaf methods available for such a purpose. Analytical electron microscopy, whether by X-ray microanalysis (XRMA) [2] or by electron energy loss spectrometry (EELS) [3,4], can now be used for the detection, quantification and mapping of nitrogen with a sensitivity acceptable for the study of bi~~logi~al samples and exceiient resolution (that of conventional electron microscopy). These methods, however, are unabte to discriminate between nitrogen isotopes. There are radioactive isotopes of nitrogen, but their half-lives are so short that they are

Correspondence

to: Dr. P. Massiot, Laboratoire “Pierre Sue”, CEA/DSM/DRECAM CE Saclay, F-Y 1191 Gif-sur-yvettt” Ccdex, France.

~1~-5~3X/93/$~6.~

of no practical use for most biological applications based on radiotracers and autoradiography. One has, therefore, to use the stable isotopes of nitrogen, 14N and “N, for labelling experiments. Natural nitrogen consists of a mixture of 14N and “N with only 0.366% 15NI but many ‘“N-enriched com~unds of nitrogen are now commercially available. Neutron capture radiography (NCR) has been successfully applied to the quantification and mapping of 14N in biological specimens [5,6], with good sensitivity and a lateral resolution in the order of 1 p.m, but this method does not detect “N. ~~~nda~ ion mass spe~trorn~t~ (SIMS) has been used for the imaging of both 14N and 15N [7,8] with a sensitivity and a lateral resolution equai, at least, to that of NCR, but cumbersome matrix effects are responsible for much of the difficulty in obtaining absolute quantitative measurements. Among the various possible nuclear processes with charged particles utilizable to detect 14N and ““N 191,WC have already proposed [IO] the USCof 3 MeV protons for the simultancous detection of “N by proton elastic backscattering spcctrometry analysis and of “N by proton induced gamma ray emission, particularly by the nuclear rcaclion “N(p n-y)‘%. In our &alysis we began the c~~rn~nta1 study of the latter possibiiity using normai or “iN-labelled samples of soybean leaves. Our aim was to use the nuclear microprobe facilities that presently exist at Bruykres-

8 1993 - Elsevier Science Publishers B.V. Ail rights reserved

466

I? Massiot et al. / Microprobe analysis of soybean leaves

le-Chatel [ll] in order to delineate the specific difficulties encountered when the analyzed matter is organic, thus preparing the more elaborate experiments which will be possible when the “Pierre Sue” laboratory’s new instruments in Saclay [12] will be available.

2. Theoretical background 2. I. 14N determinution

using PESA:

‘IN(p, p)14N

The proton backscattering method is based on both Coulomb and nuclear interaction potential with the particle and the target. This technique is sensitive in the detection of nitrogen and other light elements compared with other ion beam methods [13]. Using H ions as projectiles has several advantages. The analysed depth is greater than with any other bombarding ion, due to the smaller stopping power of the proton. The cross section for elastically scattered protons by light elements is often high, with specific pronounced resonances which strongly improve the sensitivity of the method. As shown by Rauhala [13] and Guohua et al. [14], the technique of proton backscattering in the range 1.5-3.6 MeV with nitrogen, has the advantage that the collision is completely elastic and without any nuclear reaction interference or inelastic scattering. However, there are also a few drawbacks with protons, mainly: a poorer depth resolution and a lower mass resolution than with alpha particles. The cross section of proton backscattering exhibits a deviation of the Em2 dependence of the Rutherford model, where E is the proton energy [15]. A comparison between Rutherford cross section, un, and experithat there is already a mental data, ona, indicates significant discrepancy for proton energies below 1000 keV. For example, 2 MeV protons with a detector setting at 160” with respect to the ion beam give a cross section ratio of 0.19 with ma = 17 mb and onn = 90 mb

1161. The magnitude of the deviation from Rutherford scattering depends on the energy levels of the compound nucleus formed during the collision. The strength of the resonances, corresponding to the nuclear structure: depends on the scattering angle. This complex physical phenomenon must be taken into consideration in the calculation of the scattering cross section, which is usually not well known. Most often, for proton backscattering, determination of the nonRutherford cross section is based on experiments and interpolation using mathematical fitting 1161. 2.2. “N determination

using the “N(p,

ay)“C

reaction

The proton-capture resonant nuclear reaction 15N(p, ay)‘*C is especially appropriate to “N detcnni-

nation, due to its high positive Q-value which produces a large cross section. The excited ihO nucleus which is formed decays by a-emission to the first excited level of i2C, the latter then decaying by 4.439 MeV y-emission down to the 12C ground state. To be compatible with the energy range used for 14N detection, WC have chosen to work with the high energy resonance at 2.970 MeV with a cross section of 750 mb. The 4.439 MeV y-ray characteristic of “N is easily identified through the Doppler broadening effect caused by alpha decay. 2.3. Quantitatke

analysis of 14N and “N

Nuclear methods have the advantage that they are usually very sensitive to the analysis of trace elements and they are suited to determine absolute concentrations (nitrogen contents in our present case). With PESA measurements, the 14N content of a sample, C (at./cm”), is given by the expression ‘=N,/Ij”Oj-,Eu(E,

0) dE do/S(E),

(I)

where I is the number of protons in the incident beam, N,, the number of elastically scattered protons, R the detection solid angle, u( E, 0) the cross section (which is a function of the proton energy and the angle defined by the normal to the sample surface and the detection direction) and S(E) the proton stopping power. With PIGE measurements, the “N content of a homogeneous sample of thickness t is given by the expression: C = N,/Iej;l[g(

E)/S(

E)]

dE,

(2)

where I is the number of incident protons, NY the number of y-rays issued from the nuclear reaction, E the gamma-ray detection efficiency, u(E) the cross section as a function of energy and S(E) the proton stopping power [17] of the sample. In principle, using cqs. (1) and (2) makes it possible to carry out absolute measurements of nitrogen contents. However, some of the physical parameters included in these expressions arc not known with accuracy. This is the reason why we have used standard samples for performing relative measurements. In this case, the nitrogen concentration is given by the expression: C, = C,,(S(E)s/S(E)s,)/(N,,/N,),

(3)

where C, and C,, arc the nitrogen concentrations of the sample and of the standard, respectively, S(E), and S(E),, arc the stopping powers (again of the sample and of the standard), and Iv, and N,, are the numbers of gamma-rays or of protons detected per charge unit.

In this study, the samples are so thin that the stopping power S(E) can be considered to be constant for protons penetrating the sample.

3. Experimental procedure 3.1. Apparafus The c~eriments were carried out using the proton micr~b~am line of the 4 NV Van de Graaff acccbrator at “BIuyBres-le-ChPtel” [I 11. Four rna~~~tic quadrupole lenses set up as a Russian quadruplet were used to focus the W’ beam to a spot area baling from 90 to 2% pmz. The proton beam hits the sample normal to the surface in a collision chamber. The vacuum was maintained close to 1 mPa using a turbomolecular pump. The proton energy was estimated with a degree of uncertainty of approximately 5 keV. The number of incident protons was monitored using a beam chopper placed in front of the quadrupole lenses.

100

L-!-l

120

130

170 150 160 140 scattering 'a'_oratzryc;ngie

180

Fig. 1. Measurement of the angular dependence of nitrogen sensitivity by PESA on a 0.3 pm thick polyimidc film (C,H,,N,O,) at a proton energy of 3.185 MeV.

3.2. PE The scattered protons were detected using a silicon surface barrier detector, set at 165” with respect to the ion beam (EGg Ortec: 25 mm2, resolution 14 keV) with a depleted zone thickness of 100 Fun. It was collimated to a diameter of 4 mm. Using the largest possible angle is a crucial point in this experimental approach for two main reasons. The first reason is that a large back angle provides good kinematic energy separation of adjacent masses. The second reason is that the shape of the elastic scattering excitation curves is very sensitive to the angle of scattering 1151. For the lower masses, the scattered ion dist~bution is anisotropic. Fig. 1 gives the ex~rimental angular d~~ndence of the detection sensitivity of 14N using a 0.3 pm thick ~~yimid~ foil; the advantage of work& with a large back angle in order to improve the sensitivity of nitrogen detection is clearly apparent in this figure. Our choice of 3.00 MeV or, better still, of 3.185 MeV protons (the latter being at the top of the rcsoname which improves the sensitivity of 14N but, in this case, lYN is not detected) thus appears to be suitable and sensitive enough to dcterminc 14N in plant tissues. A typical PESA spectrum of a soybean leaf is shown in fig. 2. In addition to 14N, 12C, ““C and 1601 the nucfides ‘*h4g, “IK> “%a, which are present in plants at less than 1 wt.% in dry tissues, can also be detected. 3.3. PPGE The gamma-rays issued from the “‘N(p, (uy)“C nuclear reaction were determined with an ultrapure Gc detector EGg Ortcc (50.0 x 54.4 mm”) set at 120”

with respect to the ion beam at a distance of 6.5 cm from the target and subtending a solid angle of approximately X.57 sr. The experimental detector ~csal~tion and efficiency were 2.73 keV and 20.4% at I.33 MeV (N’Co source), respectively. Fig. 3 gives the characteristic PIGE spectrum of a soybean leaf. In addition to “‘N

2068 ---__

Energy (keV) 2575

I

3085

I

I

103

102

I-

2 : 8

10'

10°

Fig. 2. PESA spectrum obtained with a 2 pm thick transversal section of a soybean leaf containing only natural nitrogen: gold coating thickness = 320 nm, proton energy = 3.185 MeV, detection angle = X5”, beam density = 15 pA/pm2, Q = 1.25 WC.

P. Massiot et al. / Microprobe analysis of soybean leaves

468

we can see that i”B > ilB 9 and a few other nuclides also be detected.

Energy (keV) 704

I

1410

2117

2623

I

3530

4236

I

can

1

4. Preparation

of the biological

samples

103

”

c

lo*

2 0

0'

10° 300

600

900

1200

1500

1800

Channels

Fig. 3. PIGE spectrum obtained with a 2 pm thick transversal section of a r5N-labelled soybean leaf: proton energy = 2.970 MeV, detection angle = 120”, beam density = 15 pA/ km’, Q = 2.20 pC.

As is the case in X-ray microanalysis by electron beams, our aim was to preserve the specimen morphology and ultrastructure by limiting the disturbance of the in vivo distribution of diffusible elements, and avoiding contamination of the preparation. With conventional chemical fixations, structures are well preserved, but soluble forms of nitrogen are lost in significant amounts. The analysis of frozen hydrated sections [18] would probably be the best way to ensure good preservation of biological structures without modifying the elemental composition existing in the living tissue; but the transfer devices for frozen sections were not available on our target wheel microprobe. Although cryosubstitution does not totally exclude some solute extraction [19], we chose to use this method which has already been claimed to be well adapted to nitrogen studies [20]. In its details, the technique was similar to that used for the preparation of plant specimens for SIMS (secondary ion mass spectrometry) analysis [8].

soybean

leaf -5-2

\

\

77

Frn thick

:: ” /

tranversal

sectlon

/

electron-microscopy double copper grid

proton

beam

sItIcon surface barrier detector (165’) ultrapure Ge detector

(120’) Fig. 4. Schematic representation

J

gold coating (320 nm thlck)

resin embedded soybean leaf

(2 pm

thick)

of the excision of a soybean leaf transversal section and the experimental arrangement.

l? Mmsiot et al. / Microprobe analysis ofsoybeun learxs

Germination and culture of the soybean plants were achieved as described previously in ref. [I], except that nitrate was supplied in the form of 1 mM KNO,. At the age of 16 days, half of the plants were transferred to a nu~ient solution ~ntaining i5N0, (99 at.% “N), and the other half were left in the basic nutrient rne~~ supplied with natural nitrogen nitrate. Fortyeight hours after the transfer, the first ~ri~oliolatc leaves from both ‘SN-labelled and control (‘“N) plants were harvested. Because plant tissues are particularly sensitive to traumatic stress, samples from the leaf laminae were excised and cryofiied as quickly as possible, according to the technique described in ref. [21]. Briefly, a stainless steel tube with a sharp circular tip (1 mm diameter) was chilled in liquid propane, and then plunged through a soybean leaf. The punched out leaf samptes were transferred into melting acetone containing 5% dimetho~~ropane [22] and activated molecular sieve 3A (Merck), and were kept in this ~lution for three weeks at -83°C [23,24]. Resin irl~ltration was car&d out after quick rewarming. The samples were pre-emhedded for 30 min in a mixture of Spurr’s resin and acetone 1: 1 (v : v) at room temperature, then they were embedded in Spurr’s resin overnight at 60°C [25]. To prevent solute losses during sectioning, the samples were dry-cut with a glass knife (Reichert Ultracut E Microtome). Two kinds of semi-thin transversal section samples were made (2 &rn thickness). The first was merely sandwiched between the two parts of an electron-micro~py double copper grid containing no other heavy material, in order to prevent too strong a ba~k~~ll~d in the scattering analysis (bar = 50 pm, lattice = 250 p,m). The second, glued to an ultrapure tantalum holder (9Y.9% purity, Goodfellow) with a

469

droplet of a mixture of 99% ethylcn glycol-I % ethanol, was subsequently dried for 30 min at 60°C then for 6-14 h at 13.5”C, and coated by a thin gold film, in order to enhance thermal and electric conduction. Fi 4 shows sch~~dtic~ly how the samples were excised and how they were mounted for analysis.

5. Results 5.1. Calibration curues

To check the linearity of the calibration curves, 0.5 pm thick pieces of nickel foil (Goodfellow) were implanted with four different doses of either “*N’ or “N*‘. The four 14N and r5N ion dose were 3.46 x IOi’, 3 46 X 1016, 7.15 X 1016 and 1.0 X 1017 ions/cm’ respectively. For 14N they correspond to 0.0~5, O.~S9~ 1.872 and 2.618 wt.%, and for ‘“N to 0.0969, ~~.9692, 2.002 and 2.801 wt.%, with a 10% variation c~~fici~~t on each value. Ion implantation was performed with 50 kcV incident ions. The TRIM code [26], based on a mathematical Monte Carlo simulation, gives a mean range of 54 nm with a straggling of 24.5 nm. In prcvious experiments [lo], we demonstrated that there was no loss of nitrogen in the nickel foils under the proton beam. The iutegratcd charge was 0.6 $2 at the point hit by the beam, with a beam area of 30 pm x 30 pm. At least three points of each standard were analyzed. The two calibraFion curves were established at the m~mum sensitivity for “N and lsN, i.e. at the top of the resonance of the two reactions, 3.185 MeV and 2.970 MeV, respectively. The reason for this choice was that the proton energy loss in the thickness of the

4oo-- ’ ’ ’ ’ ’ ’ ’ ’ r=0.996

350

300

y

1

250

/

c

V

h a 200 c i? z

J

150

100

50

b

a 5 0

IO

20

30

40 doses

50 (x

60

70

10’50t/cmZ

80 )

90

100

110

0 ~,1,1~~ 10

20

30 doses

40

50

60

70

80

90

100

110

(x 1 0’5at/cm2)

Fig. 5. Calibration curves obtained for 14N by PESA (a), and for “N by PIGE tbb):nitrogen doses ranging from 3.46 x lO= to 1 X 1017ions/cm2.

470

P. Massiol el ul. / Microprobe an&k

nickel layer implanted with nitrogen ions was less than the resonance widths. Fig. 5 shows good linearity of the number of counts per PC as a function of the concentration of nitrogen, with values of the regression coefficient of 0.993 for 14N and 0.996 for “N; the reproducibility was 2.6% for the highest nitrogen content, and 19% for the lowest.

It is well known that damage and loss of major and minor elements occur during irradiation with charged particles. This is especially the case when attempting to improve the lateral resolution by focusing the incident ion beam more finely, as is presentiy the case with most microbeam lines in the world. The current density then has to be increased, in order to preserve the statistical significance of the measurements. This results in increasing radiation damage, and may be responsible for erroneous analytical results. Cookson has reviewed this problem [27], listing the possible mechanisms causing damage: sputtering, elemental migration, heating and atomic displacement. The beam-induced loss of matter is greater on an organic rather than on an inorganic matrix, due to the low value of both the thermal and the electrical ~nductivi~ of organic matter which increases the st~ctu~l alterations. Heating effects were first described for electron microscopy by Talmon and Thomas [28], then, for the proton microprobe, by Vis [29], and more recently by Cholcwa et al. [30,31], and by McColm and Cahill [32]. According to theoretical calculations by Talmon and Thomas 1281, the tcmperatur~ rise due to conduction in an organic target irradiated with an unscarmcd beam is given by the expression: Tmax- T,, = w:[ I + 2 ln( R,/r0)l/4k,

ofsoybeanleaves

for the quantitative microanalysis of biological material under normal irradiation conditions, the mass loss was mainly de~ndent on the absorbed dose and on the total energy transferred to the target. In this methodological study of nitrogen detection in plants WCchose to lay our specimens on a polyimide film (C221~,,N,0,), whose composition is close to that of biological samples and whose behaviour under irradiation is better than that of polyester US]. In a previous study we demonstrated a stable dist~bution in time of nitrogen under the beam [lo]. In this analysis we have studied the effect of 3 MeV stationary protons, with a beam current of 2-4 nA and a spot size of approximately 250 p.m’, on 2 p_rn thick transversal sections of soybean leaves. The i~adiati~ns were carried out with 0.05 p.C/pixel, 25 times at the same point. Using cq. (4), the temperature rise was estimated to bc approximately 440 K close to the point of impact of the beam. A gold layer 320 nm thick was coated on the samples in order to render them conductive. Under such ~nditions~ it may be assumed 1351 that the temperature increase in the binlogical matrix was reduced by approximateIy 50 K, with sputtering and evaporation cffccts reduced accordingly. The dccrease of the nitrogen signal occurring during the accumulation of a charge of 1.25 p.C is shown in fig. 5. A beam density of about 15 PA/pm’ was responsible for a nit~~en loss in the range of 50-60% of its initial content, occurring mainly during the first 2 min of irradiation (i.e. up to an integrated charge of 0.4 ~0. This dramatic evolution of the nitrogen signal in our experimental configuration has to be taken into account to obtain accurate results with biological tissues. f0

,_-~“.--

IT

!----I

(4)

where T,,, - T; is the maximal temperature rise hetween the center of the beam spot and the ~llducting grid? y is the heat generation per nnit of volume, rit is the radius of the beam, R, is the separation between beam impact and the conducting grid, and k is the average specimen conductivity. To improve the conduction and radiation models, more precise information will have to be provided, especialfy concerning c(~nductivi~, specific heat, melting point and emissivity [30]. Atomic displacement and ionization are also causes of light element loss in amorphous matrices, due to the breaking of chemical bonds. But this phenomenon is not predominant compared to the effect of electronic collisions when the film thic~c~ is smaller than the ion range 1331. This was the case in our present experiments, where the film thickness was 2 pm and the proton range 105 km, Using another approach, Themner [34] has shown that,

Fig. 6. Nitrogen bchasiour in a 2 pm thick section of a soybean leaf under 3 MeV proton microhram bombardment: gold coating = 320 nm, beam density = 15 PA/pm’.

P. Massiot el uL / Microprobe ana1ysi.sofsoybean leaves

Hence, to be able to obtain reliable quantitative results for the me~urements carried out in this study, we had to perform the nitrogen analyses under impedimental conditions identical to those corresponding to the average curve given in fig. 6, and to correct for the nitrogen mass loss accordingly. Many important experimental parameters can be optimized in order to minimize the degree of chemical element loss and of structural damage [27,36]. Structural irradiation damage can he better controlled using a high spatial resolution surface analysis such as atomic force microscopy [37]. Continuous scanning analysis would reduce thermal damage, although it does not depend very much on the scanning frequency 131,343. Nevertheless, for this analysis scanning was still not available. 5.3. Experimental approach for quantitative analysis of 14N and “N in soybean leaves

In a first series of experim~nts~ t4N and 15N measurements were carried out in five different areas of a 2 pm thick section of a soybean leaf containing natural nitrogen. Then, four nitrogen measurements were performed on a 2 km thick section of a soybean leaf labelled with *‘N. The proton beam density was 15 pA/Fm2, and the integrated charge was 1.25 p,C for 14N and 2.20 p-C for rsN. Observation of the specimen structure was carried out in the target chamber using only a binocular optical microscope with a magnification of 400. The lighting system was based

on optic fibers,

and its accuracy

411

was not very good. For this reason, we were not able to determine which leaf tissue (veins, chi~ro~hyilian tissues or epidermis) in the studied hist~)l~~~ic~~l section was that actually bit by the beam and analysed for nitrogen. For the quantitative analysis of nitrogen based on relative measurements, two kinds of standards were employed for r4N: i) nickel foils irnpl~~lt~d with 14N ions at a ~n~~ntrat~~n of 0.~ wt.%, and ii) a 0.3 pm thick polyimide foil with a 100 nm thick gold coating. For r4N, WC used only nickel foils implanted with ‘“N ions at a concentration of 0.969 wt.%. For these ion implanted standards, the energy loss of a 3 MeV proton to reach the implantation profile (mean range: 54 nm, st~ggIing: 24.5 nm) was appro~mat~ly 3.2 keV in nickei. Rue to the thinness of the ion implanted standards the experiments were not carried out at 3 MeV but at the top of the resonances for t4N and “N, i.e. at 3.185 MeV (the resonance width, I’= 11 kcV) and 2.970 MeV (I’= 45 keV), respectively. The proton enloss of 3.2 keV was smaller than the resonance widths used for the analysis of 14N ‘and “N, which caused the quantitative evaluation of nitrogen contents to be underestimated when using the ion implanted standards. With the 0.3 p,rn polyimide standards this problem was not encountcrcd. The nitrogen concentrations was calculated using cq. (3). As expected from the above rationales the first qu~tita~v~ results obtained with the ion implanted standards were less than in previous data [38]. In resin-embedded sections of a leaf containing only natural nitrogen, the 14N content after nitrogen loss correction was found to be 0.91 &

Table I Uuantitative analysis of 14N and ‘“N with a 2 urn thick section of soybean leaf with nitrogen natural abundance or ‘SN-labeUed: gold coating ~“2 320 nm, beam density = 15 pA/pm2, for 14N Q = 1.25 uC, for ‘sN Q = 2.20 )LC. Concentrations corrected for the nitrogen contribution in Spur&r resin (10%) and the nitrogen mass loss (Go%)

“-1

‘“N ~n~~n~~~~~ [wt.%]

14N ~n~ntration

[wt.%1 (E, = 3.185 MeV)

2 pm thick transversal section of a soybean leaf with ‘sN in natural abundance Average 2 urn thick transversal section of a 15~-la~~I~d soybean leaf Average

*-“~

with 14N ion implanted standard

with polyimide standard

1.00 1.06 0.95 0.83 0.69 0.91+0.14

‘1.58 1.48 1.34 1.39 1.20 1.40*0.14

0.67 0.88 0.82 0.80 0.79 f 0.09

-

(En = 2.970 MeV) with lSN ion implanted standard .not detectable due to the nitrogen mass loss under the beam

0.14 0.11 0.11 0.08 0.11 f 0.03

472

P. Massiot el ul. / Microprobe analysis ofsoybean leaces

0.14

wt.%. The standard deviation was estimated from the five measured contents which are summar~ed in table 1. When using the polyimide standards, and taking into account the difference between the nitrogen mass loss and the nitrogen contribution of Spurr’s resin (the latter is estimated at 10% of total nitrogen content detected by mass spectromctry or ion microprobe), the i4N ~nccn~ation was found to be 1.40 i 0.14 wt.%. Unfo~unately, the nitrogen loss under the beam makes it impossible to measure the i5N content of samples containing natural nitrogen. For the measurements in a 15N-labelled leaf, the data were underestimated due to the use of ion implanted standards as a reference: WC found that the average nitrogen mass loss was 0.79 i 0.09 wt.% Eor 14N and 0.11 f 0.03 wt.% for t5N. There are three main reasons for the large degree of uncertainty in these results: i) the lack of a precise control of nitrogen mass loss, which can vary appreciably from one experiment to another, ii) the poor spatial resolution of the microbc~~ which made it impossible to take the tissue hetero~~nei~ into account, and iii) the fact that the thickness of the ion implanted standards was less than the resonance width of the nuclear reactions used. Despite these difficulties, the isotropic ratio “N/14N was found to be in the range 10.0-20.X% in the “N-labelled leaf, which is consistent with SIMS measurements (14-23%) on the same samples [S].

6. Conclusions In this first approach of the detection of i4N (by PESA) and ““N (by PIGE) in histological sections of soybean leaves, as carried out with the proton microprobe of Rruyi?res-le-Chatel, we have begun to assess the main experimental parameters involved in such measurements. When i4N and iSN implanted nickel foils were used as standards, there was very good linearity for the number of nitro en counts per PC as a function of the nitrogen concentration for both isotopes. With the soybean leaves, the radiation damage under the beam was very significant, amounting to a decrease of the nitrogen signal in the range SO-60% following the delivery of an integrated charge of 0.4 p,C (i.e. in the first 2 min of irradiation under our present expe~ment~ ~nditions). However, it will be possible to minimize this drawback to a large extent by better optimization of experimental characteristics, especially when the new nuclear microprobe facilities at the Pierre Sue laboratory (CEA/CNRS, Saclay, France) are available. In our present study, by correcting our data for the nitrogen loss cffcct, we have measured an average concentration of 14N equal to 1.4 wt.% by weight in soybean lcavcs containing only natural nitrogen, This is consistent with the nitrogen eon-

tent of plant samples, ranging from 1 to 3 wt.% in dry tissues, as usually accepted 1381. With “‘N-labelled plants, isotopic ratios tsN,/i”N, which were d~t~rnlined using the nuclear microprobe, were consistent with the data obtained by SIMS measurements on the same samples. These results are encouraging with regard to the possibility of achieving the simultaneous detection of ‘“N and “N at the same point on a plant sample.

Acknowledgements We are grateful to M. Mosbah, P. Trocellier, N, Toulhoat and F. Mercier for their invaluable advice.

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