STANDARDS FOR QUANTITATIVE X-RAY MICROANALYSIS OF BIOLOGICAL SPECIMENS

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STANDARDS FOR QUANTITATIVE X-RAY MICROANALYSIS OF BIOLOGICAL SPECIMENS

W. de Bruijn

To cite this version:

W. de Bruijn. STANDARDS FOR QUANTITATIVE X-RAY MICROANALYSIS OF BIO- LOGICAL SPECIMENS. Journal de Physique Colloques, 1984, 45 (C2), pp.C2-469-C2-472.

�10.1051/jphyscol:19842107�. �jpa-00223774�

STANDARDS FOR QUANTITATIVE X-RAY MICROANALYSIS OF BIOLOGICAL SPECIMENS

W.C. de B r u i j n

Centre for Analytical Electron Microscopy, Laboratory for Electron Microscopy, Rijnsburgerueg 10, 2333 AA Leiden, The Netherlands

Résumé - Il est proposé d'utiliser des particules sphériques ëchangeuses d'ions comme étalon pour estimer la proportion des divers éléments dans les tissus biologiques.

Abstract - The use of ion exchange beads is proposed as standards for the estimation of various elements in biological tissues.

Elements to be detected or quantitated in biological tissues occur in three forms:

as free ions or salts, in a crystalline phase, and bound to a matrix. For the latter category a standard system using ChelexlOO -j o n exchange beads is proposed. Cations can be attached in a controlled way to such an artificial polystyrene divinyl benzene matrix, furnished with negatively charged iminodiacetate functional groups

by replacing the sodium ions present in the commercial product.

In Fig. 1 beads measuring 35-75 ym in 0 are shown in the SEM-mode as standards for bulk analysis, attached to a graphite plate with graphite glue (Leit C ) . The analysis of Co-loaded beads is given in Fig. 2. Loading conditions from salt solutions are described elsewhere (1).

The binding is controlled mainly by the pH of the reaction. After loading, the ex- cess of the equilibrium solution can be washed away by rinsing with a buffer having the same pH as the equilibrium solution. When the beads are maximally loaded, the concentration of the various elements vary - according to the differences in affinity- between 2 and 15 w/w% (as far as we have measured them), but almost every concen- tration below the maximum value can be obtained by changing the equilibrium con- ditions. The concentration on the maximally loaded beads was determined at 40 or 60 kV on 35-75 ym 0 beads with either the pure metal or copper as a reference. On iden- tical samples the concentration was determined by neutron activation (Interuniversi- tair Reactor Instituut, Delft; Table I ) .

TABLE I. Mean maximum concentration on Chelex -beads (in X w/w).

Zn Cu Ni Co Fe Mn Ca Mg Na H(Na) Neutron acti-

vation 12.2 12.7 9.8 12.1 11.5 9.4 6.5 4.1 6.3 0.01 40 kV 13.0 13.9 12.9 10.5 11.5 10.5 6.2 3.7 — 0 + S.D. 2.3 1.6 0.6 0.5 0.6 0.5 0.6 1.8

60 kV 12.6 13.7 11.5 10.5 11.4 8.2 5.5 4.9 — 0 + S.D. 0.8 0.5 0.4 0.4 0.7 0.5 0.5 0.7

In the spectra sometimes some residual CI concentration is present in addition to the requested peak, as in most cases chlorine salts are used for the exchange. The The use of nitrates will prevent that.

Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:19842107

C2-470 JOURNAL D E PHYSIQUE

F i g . 1. Chelex beads o n a g r a p h i t e s u b s t r a t e i n SEN-mode. Bar: 100 um. I n s e t : f u n c - t i o n a l group e q u i l i b r a t e d w i t h Nat.

F i g . 2. Spectrum o f c o b a l t - l o a d e d Chelex beads 60 kV, 100 nm s p o t - s i z e . The conver- s i o n o f t h e Na i n t o Co beads i s complete. The c o b a l t c o n c e n t r a t i o n was e s t i - mated t o be: 10.5 ( o r 12.1)% ( T a b l e I).

F i g . 3. P.K.C.-program f o r measuring beads and X-ray a n a l y s i s i n t h e centre.Bar: 10 um.

F i g . 4. Ag-'1P 1-beads loaded w i t h phosphate, spectrum t a k e n a t 60 kV,100 nm s p o t p l . F i g . 5. Superimposed images o f a bead and i t s measuring by t h e P.R.C.'program, t o

demonstrate t h e accuracy o f t h e measurement. Bar: 10 um.

F i g . 6. Ag-MP 1-beads loaded w i t h bromine, which more c o m p l e t e l y removes t h e cohimer-

c i a l l y p r e s e n t c h l o r i n e t h a n t h e phosphate d i d . 60 kV, 100 nm s p o t s i z e .

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n between t h e c a l c i u m and m ne b e a d , w i t h f i v e d i f f e r e n t mutual r a t i o s , and beads w i t h o n l y c a l c i u m o r magnesium (Edax, Edit-EM r a t i o program) SEM-mode.

F i g . 8. U l t r a t h i n s e c t i o n w i t h bead-fragments c o n t a i n i n g b o t h Ca and l~lg ( r a t i o E i n F i g . 7 ) . R e s u l t s g i v e n i n Table I 1 ( s e c t i o n 3 ) .

F i g . 9. 1.P.P.-measuring program e s t i m a t i n g t h e s u r f a c e o f t h e c r o s s - s e c t i o n e d Chelex bead c o n t a i n i n g osmium. Reduced R a s t e r Program. M = 1 3 3 . 3 3 3 ~ Fig.10. R e l a t i o n between t h e determined s p e c i f i c g r a v i t y o f t h e loaded beads ( p ) ,

t h e s e l e c t i v i t y c o e f f i c i e n t ( s ) and t h e atomic w e i g h t ( A ) .

F i g . 11 P t - c o n t a i n i n g monocytic g r a n u l e s r e v e a l i n g p e r o x i d a t i c enzyme-activi ty ( r ) and 12. CO-embedded w i t h P t - c o n t a i n i n g Chelex beads 0). G l u t a r a l d e h y d e f i x a t i o n .

Unstained s e c t i o n . 80 kV. Bar: 10 pm ( 1 1 ) . Bar: 1 pm ( 1 2 ) .

C2-472 JOURNAL DE PHYSIQUE

Beads w i t h q u a t e r n a r y ammonium groups a t t a c h e d t o a s i m i l a r s t y r e n e d i v i n y l ben ene cop01 nier l a t t i c e a r e a v a i l a b l e (Bio-Kad, AG-MP 1 ) t h a t b i n d anions, such as Poi-, Br,SO $ -, and CN-, t o t h a t m a t r i x . I n s p e c t r a of such beads a g a i n some r e s i d u a l c h l o - r i n e 4s observed b e i n g t h e commercial c o u n t e r i o n , which i s n o t com l e t e l y removed.

Sodium, t h e commercial c o u n t e r i o n o f t h e n e g a t i v e l y charged ChelexBoO bead, i s a p p a r e n t l y n o t d e t e c t e d due t o t h e bad d e t e c t i o n c o n d i t i o n s f o r t h a t element w i t h EDS d e t e c t o r s ( F i g s . 4 and 6 ) . The AG-MP-1 beads t o w h i c h a n i o n s a r e a t t a c h e d have n o t y e t been t e s t e d by a r e f e r e n c e system, p a r t i a l l y aue t o t h e f a c t t h a t p u r e e l e - rnents a r e h a r d t o o b t a i n t o p e r f o r m t h e same X-ray m i c r o a n a l y t i c a l d e t e r m i n a t i o n , p a r t l y due t o t h e f a c t t h a t we have n o t y e t been a b l e t o remove t h e c o u n t e r i o n C1 c o m p l e t e l y .

Besides i t s use as s t a n d a r d i n c o m b i n a t i o n w i t h b u l k specimens, such beads can be used as t e s t o b j e c t s f o r a u t o m a t i c measuring-programs, i n t e g r a t i n g morphometry and X-ray a n a l y s i s . Such as t h e T r a c o r N o r t h e r n Program P.R.C. ( F i g s . 3 and 5 ) . A l s o two elements can be a t t a c h e d t o t h e bead m a t r i x a t t h e same t i m e . We o f f e r e d ~ a 2 + and ~ g z + - i o n s t o known amounts o f 20-30 um fi beads i n a t o t a l c o n c e n t r a t i o n o f 0.54 M, and v a r i e d t h e mutual m o l a r r a t i o between Ca and Mg f r o m 1:5, v i a 3:3 t o 5.1, and measured t h e elemental Mg/Ca r a t i o . The r e g r e s s i o n c o e f f i c i e n t was 0.99 ( F i g . 7 ) . F o r t h e b i o l o g i c a l specimens t o be observed i n t h e CTEM-mode t h e beads a r e e x c e l l e n t f o r co-embedding w i t h t h e t i s s u e t o be analysed.

To embed t h e beads, t h e y were added d i r e c t l y t o t h e f i n a l monomer c o n t a i n i n g t h e t i s s u e t o be i n v e s t i g a t e d , as a d r y m i l l e d powder o r soaked w i t h epoxy propane. I n F i g . 8 such u l t r a t h i n s e c t i o n e d beads a r e shown. I n t h i s case such beads were used t o t e s t t h e homogeneous d i s t r i b u t i o n o f t h e elements Ca (and Mg) by comparing 5 p o i n t a n a l y s e s i n each c r o s s - s e c t i o n e d bead i n d i f f e r e n t s e c t i o n s ( T a b l e 11). A1 though t h e SD v a l u e s a r e l a r g e r t h a n t h o s e f o r t h e v a l u e s i n Table I, due t o t h e r e s t r i c t e d analysed volume i n t h e c r o s s - s e c t i o n s , i t can be c o n s i d e r e d a c c e p t a b l e f o r a commer- c i a l p r o d u c t , n o t made f o r such a p r e c i s i o n . I t can b e observed t h a t t h e Mg values measured i n t h e s e u l t r a t h i n s e c t i o n s approach t h e minimal d e t e c t a b l e l i m i t o f t h e system; whereas t h e v a l u e s i n t h e b u l k specimen were e a s i l y d e t e c t a b l e ( F i g . 7, E).

To demonstrate t h a t v a l u a b l e parameters f o r X-ray a n a l y s i s can be o b t a i n e d w i t h t h i s system, we determined t h e d e n s i t y o f t h e (35-75 um fi) beads i n t h e vacuum-dried form, l o a d e d w i t h 12 a s s o r t e d c a t i o n s and t h e unloaded H+ form. A f t e r l o a d i n g and c a r e f u l washing a l i q u o t s o f t h e vacuum-dried beads were c a r e f u l l y weighed and, u s i n g a C o u l t e r Counter system, t h e number o f beads was determined. By measuring t h e mean c i r c u m f e r e n c e o f s e v e r a l hundred l o a d e d beads i n SEM micrographs by u s i n g an X-Y t a b l e t , t h e i r mean d r y volume was determined. The d e n s i t y values were w i t h i n t h e r a n g e g i v e n by o t h e r s ( F i g . 10).

MEAN Ca AND Mg COUNTS I N ULTRATHIN SECTIONED BEADS/lOOs T a b l e I 1 Counts Ca ( K a ) S.D. ( % ) Mg (Ka) s i g n i f i c a n c e s e c t i o n 3 ( n = 26) 60 kV 3885

s e c t i o n 4 ( n = 21) 3854 s e c t i o n 4 ( n = 21) 80 kV 2581

s e c t i o n 5 ( n = 25) 3337 9.5 9'

Q u i t e r e c e n t l y such c r o s s - s e c t i o n e d beads a r e used as a t e s t - o b j e c t f o r t h e semi- a u t o m a t i c 1.P.P.-analysis programs, f o r t h e a c q u i s i t i o n o f b o t h t h e s u r f a c e and t h e mean c o n c e n t r a t i o n o f elements i n c e l l o r g a n e l l e s i n u l t r a t h i n s e c t i o n s ( F i g . 9 ) . The presence o f , i n t h i s case, P t - c o n t a i n i n g Chelex beads between a c r o s s - s e c t ~ o n e d c e l l p o p u l a t i o n w i t h P t - c o n t a i n i n g r e a c t i c n p r o d u c t s f r o m a cytochemical r e a c t i o n i s shown i n F i g s . 11 and 12.

References:

1. De B r u i j n , W.C., (1981) I d e a l s t a n d a r d s f o r q u a n t i t a t i v e X-ray m i c r o a n a l y s i s o f

b i o l o g i c a l specimens. SEM/II , 357-367 (SEM 1nc.AMF O'Hare Chicago, I L 60666, USA).