localization of mineral elements in normal and strontium-intoxicated quail eggshell by secondary ion...

Calcif. Tissue Int. 30, 151-161 (1980) Calcified Tissue International ,~ 1980 by Springer-Verlag

Localization of Mineral Elements in Normal and Strontium-Intoxicated Quail Eggshell by Secondary Ion Mass Spectroscopy and Electron Probe Microanalysis

Carmen Quintana,*, ** Annick Quettier,* and Daniel Sandoz**

*D6partement de Biophysique, Faculte de Mddecine de Cr6teil, 8, rue du G6n6ral Sarrail--94000 Creteil, France; and **Centre de Cytologie Exp6rimentale du C.N.R.S., 67, rue Maurice GOnsbourg, 94200 Ivry sur Seine, France

Summary. Localization of mineral elements in the shell of quail eggs was studied by secondary ion mass spectrometry (SIMS) and electron probe microanalysis (EPMA). Normal eggs as well as eggs from hens intoxicated with strontium were studied.

A simple preparative technique was employed that is advantageous for the study of mineralized tissues. Semithin (1 /zm) sections of material embedded in Araldite were used. These preparations allow analysis by both SIMS and EPMA, and do not exhibit charge distortion in the resulting ion images.

The greater sensitivity for detection of elements in low concentration and greater statistical reliabil- ity of images with SIMS allowed us to show the stepwise variations in concentration of Mg within normal and strontium-intoxicated quail eggshell. These steps were not detectable with EPMA.

Strontium chloride added to the food was incor- porated as Sr into the eggshell, which was abnormal in development. The incorporation of Sr does not correspond to the calcium distribution. The cone layer appears less rich in Sr, and it is also less rich in Mg in normal as well as in intoxicated eggs. From this distribution it is possible to construct a diagram of the abnormal eggshell which is difficult to distinguish by other methods. Electron diffraction of the quail eggshell intoxicated with Sr showed the carbonate to be in rhombohedric rather than ortho- rhombic form, even though the strontium concentration may be very high. These findings are a re- flection of a complex process in dynamic equilibrium which is responsible for the biomineralization of the eggshell of quail.

Key words: Electron probe microanalysis - - Sec- ondary ion microanalysis - - Quail eggshell - - Biomineralization -- Elemental distribution, St, Mg, Ca.

S e n d ~:~(fprint reques t s to C. Quintana at the above address.

Introduction

The eggshell of birds is an advantageous biological model for studying the distribution of mineral elements by microanalysis since it is approximately 95% mineralized. It is possible to compare directly the distribution of elements by microanalysis and data on the ultrastructure and crystallography of this material by electron microscopy. In a previous article [1] we have described the ultrastructure and the distribution of Ca, Mg, P, and S in quail eggshell. These results were obtained: (a) on thin sections of material by electron microscopy and electron diffraction or (b) on semithin sections (approximately 1 # m thick) observed with the light microscope and studied by electron probe microanalysis. All preparations were undecalcified and embedded in Araldite,

Calcium carbonate, as calcite crystals, constitutes the mineral of the shell. This calcite contains magnesium which is partitioned in a nonuniform fashion in the different layers of the shell [1, 2]. Oth- er cations such as strontium are often associated with biological calcium carbonate [3], but they were not detected in the shell using electron probe microanalysis. The greater sensitivity of secondary ion mass spectroscopy compared to EPMA [4] has led us to use this newer method to study the distribution of magnesium in the different layers of the shell and to confirm the absence of strontium in the normal egg, within the limits of sensitivity of the method.

We have also sought to introduce strontium into the calcium carbonate of the egg shell by intoxica- tion of the quail with strontium chloride, in order to study the incorporation and partition of this element in the shell.

In this article we compare the analytical results obtained by SIMS and EPMA in the normal quail eggshell and the shell intoxicated with SrCI=,. The

0171-967X/80/0030-0151 $02.40

152 c . Quintana et al.: Localization of Elements in Quail Eggshell by SIMS and EPMA

u l t r a s t r u c t u r e a n d c r y s t a l l o g r a p h y o f t he s h e l l en -

r i c h e d in s t r o n t i u m c o m p l e t e t h e r e s u l t s .

Material and Methods

Japanese quail (Coturnix coturnixjaponica), raised at 24~ with 14 h light and laying regularly each day, were fed with husked rice with added SrCI2 (20 g/kg). Laying ceased immediately after beginning treatment.

One quail was sacrificed after 6 days of treatment. The small egg which was removed from the uterus had a cuticle rich in green pigments. It was this egg which was studied. The eggshells from nontreated quail that received a complete diet were used as standards.

Fragments of shell were fixed with 4% glutaraldehyde in re- tonal buffer at pH 7.2. After rinsing with buffer, the specimens were sometimes post-fixed with 1% OsO4 in buffer. After dehy- dration in ethanol, the pieces were embedded in Araldite [1]. Semithin sections were cut on an ultramicrotome with a glass knife and thin sections were cut with a diamond knife.

Shell as all strongly mineralized systems is a poor electrical conductor. In order to avoid charge accumulation in both methods of analysis, the surface of the specimen is made conductive. For EPMA we used semithin sections (ca. I ~m thick) deposited on a plastic support of low average atomic number (Terphane) to reduce the X-ray continuum as much as possible. The sections were then coated with a layer of carbon [5]. For SIMS the specimens were usually prepared as ground sections of a few tens of microns in thickness, and the surface polished by means of diamond paste until a metallographic polish was obtained (about 0.5 /xm) [4]. Finally, to conduct the electric charge accumulated on the surface during analysis, a grid of aluminum was deposited on the surface [4, 6]. Later, in order to simplify the specimen preparation we utilized semithin sections of the same thickness used for EPMA and deposited them on platinum supports, as is done for examination of histological sections [7].

The X-ray images of Ca, Mg, and Sr were made with an acceb crating voltage of 15 kV in a scanning electron microscope equipped with X-ray wavelength dispersive spectrometers. To obtain the X-ray images of Ca. we used a current of 10 to 20 hA. For Mg and St, the current was 50 to 75 nA.

The mass spectrum and ion images were obtained with a SIMS apparatus, in which the mass resolution is 300. using methodology that was previously described [4, 8]. It is possible to obtain direct images of the distribution of elements which differ from one specific unit mass. However, for several specific unit masses, interfering ions of different exact mass may exist. For example, in our case, we found at nominal mass 24 the ions Mg + and C2 +, which have an exact mass of 23.985045 and 24.00000 atomic mass units, respectively. With the apparatus we used it is impossible to distinguish the contribution of the mass inter- ference in the image of a given mass. But when this apparatus is equipped with an electrostatic analyzer, one can obtain mass spectra with a resolution up to M/AM of about 3000 (M is the mass number and AM is the difference between the exact masses of the two ionsl. This allows one to separate certain interfering peaks such as C, + and Mg §

EPMA is done throughout the thickness of the section and is not destructive, whereas SIMS is done by successive destructive sputtering of the section.

The ion images give a surface resolution better than 1 v.m for a circular field of 250 v.m diameter at a magnification of 100. The depth resolution is the order of 50 at 100 A. Thus spatial resolu-

tion of the ion images is always better than that of scanning X-ray images, which is limited by the scattering of primary electrons within the sample.

Images at greater magnification may be realized with a supple- mentary device called "'transfer optics" [9]. This system is made of an assembly of electrostatic lenses which replace the immer- sion lens of the ~5IMS apparatus. It permits selection of a smaller surface of the specimen for analysis and formation of an ion image without loss of signal.

Study of the variation in emissivity of an element in a sample across a direction may be done by line scanning. In the case of EPMA, the specimen is immobile and the scanning system permits recording of variations of the X-ray signal in a continuous fashion. In the case of SIMS, the specimen is displaced at a con- stant speed in front of a circular diaphragm of 60 p,m diameter which delimits the analyzed surface. The variation of the number of ions in the selected mass is recorded along this line scan.

Results

N o r m a l Quail Eggshe l l

T h e e g g s h e l l o f b i r d s is f o r m e d by a n a r r a y o f co l -

u m n s h a v i n g the m a m i l l a r y k n o b s as t h e i r b a s e . T o -

g e t h e r t h e y c o m p r i s e t h e m a m i l l a r y l a y e r (F ig . 1).

T h e i n t e r n a l a n d e x t e r n a l l a y e r s o f c o n e s a r e n e x t

p u t in p l a c e , t h u s p e r m i t t i n g t h e she l l to b e c o m e

c o n t i n u o u s . A f t e r w a r d s , t h e p a l i s a d e l a y e r is la id

d o w n a n d o c c u p i e s 70% o f t he t o t a l t h i c k n e s s o f t he

she l l . T h e s u p e r i o r p a r t o f t he p a l i s a d e l a y e r is o c -

c u p i e d by t h e i n t e r n a l a n d e x t e r n a l supe r f i c i a l c r y s -

tal l a y e r s a n d the c u t i c l e . T h e l a t t e r is c o m p o s e d o f

o r g a n i c m a t e r i a l a n d n o n c r y s t a l l i z e d m i n e r a l s .

T h e m a s s s p e c t r u m o f t h e n o r m a l she l l is s e e n in

F i g u r e 2a. Th i s s p e c t r u m is d i f f e r e n t i a t e d f r o m t h a t

o f m i n e r a l c a l c i t e b y the p r e s e n c e o f (a) M g at

m a s s e s 24, 25, a n d 26 ( the n a t u r a l i s o t o p i c a b u n -

CU

escl

iSCl

Fig. 1. The following abbreviations are used in the figures: ca. cavity; cl, cone layer: cu, cuticle: eel, external cone layer: escl, external superficial crystal layer: icl, internal cone layer: iscl, internal superficial crystal layer: ml, mamillary layer;p/, palisade layer: sin, shell membrane.

Scheme of different layers of normal quail eggshell (see ref. 1). The thickness of the shell is approximately 200/xm

C. Quintana et al.: Localization of Elements in Quail Eggshell by SIMS and EPMA 153

I n t e n s i t y

C

I I . t0 2 0

'Na

I Cil , I

Ca

I �9 l | l l l l l l i : . , l . . I =

3 0 5 0

!],, 4 0

CaO

CaOH

,~J ,, I 6 0 7 0

i i 8 0 9 0

2 a

C a 2 0

1 0 0 M a s s

I n t e n s i t y

CH

il, l 10 2=0

( 2

3~0 40

2 b

Ca I I ( )H

C IH2Ca

, 1,1,1 l,], , , , 1 , z 5b e'o ro 8b 'gb

C a 2 0

1, 1 0 0 M a s s

Fig. 2. Mass spectra of eggshelh la) normal. (bl intoxicated ~ith strontium. Conditions are described in the text. Primary ions are O., +

dance is 78.70c;~, 10.13~, ~, and 11.17%): (b) K at mass 39: and (c) the reversal o f the relative ampli- tude o f the peaks at 56 (Ca O*) and 57 (Ca OH*) mass [10]. As indicated above , at mass 24, C2 + may be super imposed on Mg +.

H o w e v e r , w h e n we take into account the weak ampl i tude o f mass 12 (C +) which has an ion yie ld greater than that o f C., * we conc lude that Mg * is pract ical ly the on ly ion present at mass 24. This suppos i t ion was conf irmed using the high mass res-

154 C. Quintana et al.: Localization of Elements in Quail Eggshell by SIMS and E P M A

Fig. 3. Distribution of calcium and magnes ium in normal quail eggshell. Compar i son of the X-ray images and the ion images. A X-ray image of Ca; l0 nA electron beam current; 320 s exposure t ime. B X-ray image of Mg; 50 nA electron beam current; 640 s exposure time. C Ion image of mass 40- exposure time l0 s. D Ion image of mass 24; exposure time 600 s; • 275. Original enlargement of the ion images is 100• E, F Ion images of mass 40 and mass 24 made with a SIMS ins t rument equipped with t ransfer optics and a channel plate. The region analyzed cor responds to the layer of cones (bottom ~ and to a part of the palisade layer. The arrows indicate the variation in steps of Mg concentrat ion in the palisade layer, x860. Magnification of the original is 200x

olution available with the electrostatic analyzer (Fig. 4). Sodium (mass 23) cannot be considered as a natural element of the shell since it was present in the fixation buffer.

The ion image of mass 40 confirms the results obtained previously with X-ray images of Ca (Fig. 3A and 3C). Calcium exhibits a homogeneous distribution of concentrat ion in the calcitic layers and a diminution of concentrat ion at the level of the cuticle. In contrast , the ion images of mass 24 exhibit complementary information on the distribution of

Mg. In X-ray images of Mg (Fig. 3B), one observes a diminution of the concentrat ion of this element in the cone layer and then a continuous gradient at- taining a maximum in the superior part of the palisade layer. In the ion image (Fig. 3D), the diminution of the concentrat ion in the cone layer is clearer. Fur thermore , the ion image allows us to show that there are stepwise variations in Mg concentration in the palisade layer. Ion image at higher magnification permits us to see more clearly the Mg concentration steps in a region where the concentrat ion of


3H

Fig. 3. G, H Line scan of Mg, X-rays and ion signal, in the shell. The reinforcement of emission at the level of the cuticle corresponds to a charging phenomenon (see textl. The probable concentration was figured by the hatched lines

Ca is perfectly homogeneous (Fig. 3E and 3F). In order to test by another method the evidence

of step changes of Mg concentrat ion, we recorded a line scan of Mg along the different layers of the shell using EPMA (Fig. 3G). The recording did not agree with the information in the ion image. However , when a line scan in Mg is done with SIMS (Fig. 3H), one observes the same steps as in the ion image. The reinforcement seen in the external part of the shell may be explained by the presence of enhanced emissivity at the level of the cuticle, a phenomenon often observed in SIMS images. It is a charging phenomenon due to the difference in conductivity between this layer, composed almost exclusively of organic material, and the calcified layers.

S t ron t ium-In tox ica ted Quail Eggshel l

Serial semithin sections of shell were utilized for the EPMA and SIMS. In order to have a structural image, the sections were observed in the light microscope before carbon deposition (Fig. 5a). The thickness of the shell is very irregular (from 25 to 100 p,m) and less than that of a normal shell (about 200 gml . The cuticle occupies about 8057/k of the thickness of the shell in the thin area (25 p,m) and about 8% in the thicker area (100 p,m).

The mass spectrum of elements in this eggshell is seen in Figure 2b. As for the normal shell, the spectrum corresponds to a field of analysis of 250 p,m diameter. "Faking into account the diminished thickness of the shell, the field of analysis now contains, in addition to the shell, the shell membranes and part of the embedding medium. The spectrum dif- fers f rom that of the normal shell by the presence of Sr at masses 86. 87, and 88 (natural isotopic abun- dance: 9.867/r, 7.02~74, and 82.56~,~). In addition, the carbon chiefly present as mass 12 (C+), 13 (CH+), 14 (CH.,+), and 66 (CaC.,H2 +) is far from being negligible. Thus one must take into consideration the participation of C2 ~ at mass 24. We have examined the spect rum at high resolution in order to separate

the contribution of C2 ~ and Mg + at mass 24. In Fig- ure 4, the spect rum of a 60 ~ m diameter area of (a) the shell, (b) the shell membrane , and (c) the embedding medium is shown. One observes that in the shell only Mg + is present. In the shell membrane , there is emission of two ions. The embedding medium lacks Mg +. The ion image of mass 12 confirms this result (Fig. 5b). The only regions which emit carbon are the shell membrane , the cuticle, and the embedding medium (Araldite).

The distribution of Ca, Mg, and Sr were obtained in the same section with each of the methods utilized. The localizations obtained with the two methods of analysis are similar (Fig. 6a to 6f). Calcium is distributed in a homogeneous fashion in the total thickness of the intoxicated shell as in the normal shell (Fig. 6a and 6b). The partition of the concentration of Mg and Sr is unequal in the different regions of the shell.

By analogy with the results obtained with the normal shell, the partition of the concentrat ion of Mg

Intensity

4a 4b 4c

SHI~ LL M I~,1 B R ~ N tl S ARALDI~E

Mg ('2 C2

~ _ _____ .~ . M~-Ss

Fig. 4. High-resolution mass spectrum at mass 24 (M ..XM 1500) in quail eggshell. The spectrum of normal and Sr-intoxicated quail egg: (a) shell, (bl shell membrane, and (cl embedding medi- tim tAraldite). Two levels of intensity are represented

156 C. Quintana et al.: Localization of Elements in Quail Eggshell by SIMS and EPMA

Fig. 5. Quail eggshell intoxicated with Sr. a Light microscopic image of a semithin section of shell embedded in Araldite. The thickness of this shell is irregular, 25 to 100/~m. On the bottom, the shell membranes. On the top, the cuticle demonstrating variable thickness. x270. b Ion image of mass 12 (C*). Only the shell membrane, the cuticle, and the resin emit this at mass. x300.

was utilized to establish a scheme of organization of the quail eggshell intoxicated with strontium (Fig. 6g). The weakest concentrat ion of Mg is assigned the location of the cone layer. The analyzed sections contain three mamil lary knobs (mr, m2, m3), of which the dimensions are of the same order as those of the normal shell. The cone layer is slightly thicker whereas the palisade layer is hardly developed. Howeve r , SIMS shows two steps in the Mg concentration identical to that described for the normal shell (Fig. 6d). Insofar as Sr is concerned the distribution is similar to that of Mg because the cone layer is a lways less rich. There is a great difference in Sr emission between one column and another. Cer- tain mamillary knobs were rich in Sr and others were poor. When the mamil lary knobs were poor in Sr, all the rest of the columns were also depr ived of Sr with the exception of the superior part of the palisade layer where Sr is always very concentrated.

Observat ion by transmission electron microscopy of thin sections of this Sr-rich shell shows a num-

ber of differences compared to the normal shell. For example , it is difficult to recognize the morphology of the layers except for the mamillary layer (Fig. 7a), where one sees in the interior of the mamillary knob cavities filled with an organic-rich material containing small crystals of calcite (Fig. 7a) as in the normal shell [I]. In addition, above the mamillary layer there are several regions of irregular forms which are variably filled with vesicular holes (Fig. 7b).

Electron microdiffraction of the different layers always shows a pattern corresponding to the rhom- boedric symmet ry of calcite. The pattern of Fig. 7b' obtained with the aid of a goniometer stage is taken f rom a crystal of the superior part of the palisade where the concentrat ion of Sr is higher. It corresponds to the orientation (00.1) where one observes third-order symmet ry , characteristic of calcite. As in the normal shell, the cuticle (Fig. 7c) contains a large number of electron-dense granules in the absence of staining. Moreover , one finds in the basal region an unusually large number of gran-

C. Quintana et ~tl.: Localization of Elements in Quail Eggshell by SIMS and EPMA 157

Fig. 6. Distribution of Ca, Mg. and Sr in a quail eggshell intoxicated with St. Comparison of the X-ray images and the ion images. Serial sections of semithin sections (I ,urn in thicknessJ, a X-ray image of Ca: electron beam current 25 nA; exposure time 320 s, x270. b Ion image of mass 40: exposure time 10 s; x300. e X-ray image of Mg; electron beam current 75 nA: exposure time 1920 s: x 270. d Ion image of mass 24: exposure time 600 s: x 300. e X-ray image of Sr; electron beam current 75 nA: exposure time 960 s: x 270. f Ion image of mass 88: exposure time 360 s; x300

ules containing a very dense material arranged in concentric layers (Fig, 7d) which resemble spherocrystals described in the excretory organs of insects El t]. EPMA done in thin sections showed that they contained variable quantities of Ca, Sr, Mg, S, and P.

Discussion

The results concerning the localization of mineral elements obtained in the quail eggshell by SIMS of by EPMA are comparable.

From the point of view of methodology, we have

shown that semithin sections of shell embedded in Araldite permit ion analysis without presenting charging phenomenon which significantly impedes analysis of spectra or of images. This method of preparation is simpler than that utilized until now for mineralized tissue.

However, there is a problem at times with the question of artifacts due to a possible difference in emission of layers rich in organic material (such as cuticle and shell membranesl as compared with the crystalline layers of the shell. It was necessary to compare the results obtained with EPMA and SIMS in order not to confuse "'greater emission" with

158 C. Quintana et al.: Localization of Elements in Quail Eggshell by SIMS and EPMA

cuticle

6g m e m b r a n e s

Fig. 6. g Scheme of the organization of layers of this eggshell based on the information furnished by the ion images at mass 24. The mamillary layer and the cone layer have dimensions comparable to those of the normal shell. The palisade layer is barely developed

'~greater concentration." We have overcome this problem by looking for experimental conditions which permit elimination of charging phenomenon. Thus, actually, we were able to confirm that semithin sections of mineralized material in Araldite can be analyzed by SIMS without treatment other than placement on a metallic support of a pure material of high mass, for example, Pt or Au. By comparison with EPMA, SIMS has the principal advantage of a greater sensitivity of detection for elements in low concentration and with a high secondary ion yield. This is the case with Mg and Sr. This greater sensitivity (1 ppm as compared to 100 ppm by EPMA) [12, 13] is due to the absence of background noise.

On the other hand, the images made using SIMS are of higher quality than scanning X-ray images because the number of secondary ions emitted per pic- ture element is much larger than the number of characteristic X-ray photons. This fact accounts for the considerable reduction of statistical noise in the SIMS image.

Mg is weakly concentrated in the shell. In the chicken eggshell its total concentration is estimated at 1% [14]. The greater sensitivity and better statistics of ion images have allowed us to obtain evidence of stepwise variations in Mg concentration in the two types of shells, whereas X-ray images give a continuous gradation. This characteristic also permits recognition of different layers in Sr-intoxicated quail eggshell which are not apparent in thin sections examined by transmission electron microscopy. In this case, the better spatial resolution of ion images does not give better analytical results because the width of the zones in which the Mg con- centrations are different is much larger than the s u r -

face resolution. Furthermore, our samples are homogeneous throughout the thickness of the section.

Scanning X-ray images and ion images of Sr distribution give a similar surface resolution. The advantage of the ion images lies in the reduction of the required exposure time by a factor of 2.5 and in the better image statistics.

Insofar as Sr is concerned, this element is incor- porated into the eggshell when added to the quail diet. The interpretation of the heterogeneous distribution of Sr in the eggshell after 6 days of treatment suggests a number of hypotheses:

1. The calcium transport system of the uterus can accept a high level of Sr since experiments with mixtures of the s9Sr and 4~Ca showed that the bar- rier between the blood and the forming shell has a Sr/Ca discrimination ratio of only 92% [15]. 2. Mineral salts are absorbed with food during the day. A portion of the calcium (or Sr in this case) is deposited in the bone and constitutes a reserve for shell formation during the night. The bird does not eat during the night, yet the shell is deposited almost totally during this time [16, 17]. Thus it is possible that the mammillary knobs,

rich in Sr (as m2, Fig. 6g), will be formed in part from a proportion of Ca and Sr as provided in the diet and from a portion stored in the bone at the beginning of the night. Afterwards, a second nucle- ation during the night will allow the formation of smaller mamillary knobs such as ml and m3 (Fig. 6g), as well as the successive formation of cone regions, c~, c2, c3. The sole source of Ca .-'+ and Sr '-'+ would be the bone, where there is a diminution of the concentration of Sr. In the morning the animal again eats and the palisade region is formed in part by the contribution of Ca and Sr, which comes from both the food and the bones. A higher level of Sr is present in the food, and the Sr concentration is enhanced. This dynamic equilibrium between shell and bone with regard to Ca and Sr is complicated, and the above hypotheses are speculative and re- quire further experimentation. An approximate evaluation of the total concentration of Sr has been made by means of the mass spectrum. This concentration could be of the order of 5000 ppm, a high level of Sr for a calcite. The concentration of Sr within biological calcite ranges on average from 1500 to 2000 ppm in foraminifera and echinoderms, from 2000 to 2400 ppm in red algae and alcyonari- ans. This concentration reaches 2900 ppm in the bryozoan Retapore pacifica [18]. With higher concentration of Sr, the calcium carbonate crystallizes as aragonite.

In foraminifera it was shown that the Sr distribu-

C. Quintana et al.: Localization of Elements in Quail Eggshell by SIMS and E P M A 159

Fig. 7. Eggshell of quail intoxicated by strontium. Thin sect ions embedded in Araldite, t, nstained and observed with the t ransmiss ion electron microscope, a Mamillary knob of the mamillary layer ~i th a cavity filled with organic material and small crystals. • a ' Diffraction of an area corresponding to the center of the cavity, b Superior part of the shell and cuticle. The densi ty of the vesicular holes is heterogeneous. • 1900, h' Diffraction of an area corresponding to the superior part of the palisade layer. It presents the tertiar~ symmet ry which is characterist ic of calcite orientation (00. l ). r Cuticle showing a large number of e lectron-dense granules in the absence of staining. • d Detail of the two granules shown in Fig, 7c containing a material organized in concentr ic layers (spherocrystals) . • 22,500

160 c. Quintana et al.: Localization of Elements in Quail Eggshell by SIMS and EPMA

t ion is not h o m o g e n e o u s : layers rich in Sr and poor in Mg a l te rna te with layers r ich in Mg and poor in Sr

[19]. The au thors had suggested that this zone-l ike s t ruc ture could c o r r e s p o n d to an a l t e rna t ion of a ragoni te layers with calci te layers . This hypo thes i s has not b e e n suppor t ed by recen t analysis with Ra- man spec t roscopy us ing a laser microprobe [20]. This s tudy has shown that all the layers (poor or rich in Sr) were c o m p o s e d of calcite. Thus it is possible that biological calcite can admit higher Sr concen t r a t ions than those usua l ly g iven in l i tera ture . It is not surpr is ing therefore that our values should ex- ceed those found in the l i tera ture that were ob t a ined by total chemica l ana lys is .

The shell is essent ia l ly c o m p o s e d of ca rbona t e s , bu t it is not poss ib le to ob ta in an ion image of the mass 12 of these ca rbona te s . We referred above to the difference b e t w e e n emis s ion and c o n c e n t r a t i o n in the case of SIMS. It is c learly es tabl ished [4] that the ion yield of an e l emen t is a func t ion of the chem- ical b o n d i n g in which that e l emen t is engaged. Thus we have obse rved (Fig. 5b) that the ion yield of C + in ca lc ium ca rbona te is negligible compared to the ion yield of the same ion in the layer rich in organic mat te r (cuticle, shell m e m b r a n e , and e m b e d d i n g medium) .

This resul t may be used to dis t inguish the organic web of a minera l ized t i ssue , or the track of minera l - izat ion in an organic t issue.*

A n o t h e r obse rva t ion is that the mass spec t rum of the n o r m a l shell and the Sr-rich shell has a peak of 56 + (CaO +) which is less than the peak at 57 + (CaOH+), con t ra ry to that obse rved in calci te of a - n o n h y d r a t e d " minera l . In fact, when the calci te of a minera l origin is s t rongly hydra ted , one is able to obse rve an ion yield of the po lyca t ion C a O H § greater than the po lyca t ion CaO § (A. H a v e t t e - L e d e b t , pe r sona l commun ica t i on ) . G iven that the eggshell con ta ins abou t 2% of organic material , we th ink that the h y d r o g e n prov ided by the organic mater ia l plays a role here s imilar to that of hydrogen in the wa te r of hydra t ed calcite. Again , we have g iven ev idence [21] for a t r ans fo rma t ion of CaCO3 ~ C a O H u n d e r the e lec t ron beam dur ing obse rva t ion of thin sec- t ions in the t r ansmis s ion e lec t ron microscope . It is possible that , unde r ion b o m b a r d m e n t , a reac t ion of this type occurs on the surface of the semi th in sec- t ion of the shell. C a O H thus formed may e n h a n c e the in tens i ty of the peak at mass 57.

Acknowledgments. This work is part of the activities of the Biomineralization Group, coordinated by Dr. R. Lefevre, who we thank for his advice and daily encouragement. The microanalysis experiments were done at the Service of Microanalyse of the Facult6 de Medecine at Creteil directed by Professor P. Galle and supported by C.N.R.S. and I.N.S.E.R.M.

The ion images at greater magnification were done with the SIMS instrument of the Department of Solid Physics of the Fac- ult6 des Sciences d'Orsay equipped with transfer optics and a channel plate. We are grateful to Professor G. Slodzian who has permitted us to use this instrument and to Mr. R. Dennebouy for his experimental collaboration. We thank Dr. M. Truchet as we have profitted from this long experience in SIMS. We also thank Mr. M. Louette for the photographic reproductions and Mrs. M. H. Valero Garcia and C. Hissler for typing the manuscript.

We are grateful to Dr. Margaret Burns-Bellhorn who has trans- lated and corrected this manuscript.

*Note added in proof. The better results as recently been obtained with the ion images of negative secondary ions at masses 24 (C._,-) and 26 (CN-) Queltier A., Quintara C.: Dis- tinction between organic and inorganic carbon in calcium carbonate materials by secondary ion masses spectroscopy (SIMS); C,R. Acad Sci. [D] (Paris) 289:433-36, 1979

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Received Januao' 29, 1979 / Revised July 19, 1979 / Accepted July 31, 1979

localization of mineral elements in normal and strontium-intoxicated quail eggshell by secondary ion...

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