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QUANTITATIVE X-RAY MICROANALYSIS OF CALCIUM IN AN OSMIUM-TREATED BIOLOGICAL
SPECIMEN
S. Blaineau, J. Amsellem, G. Nicaise
To cite this version:
S. Blaineau, J. Amsellem, G. Nicaise. QUANTITATIVE X-RAY MICROANALYSIS OF CALCIUM
IN AN OSMIUM-TREATED BIOLOGICAL SPECIMEN. Journal de Physique Colloques, 1984, 45
(C2), pp.C2-463-C2-464. �10.1051/jphyscol:19842105�. �jpa-00223772�
30URNAL DE PHYSIQUE
Colloque C2, supplément au n°2, Tome 45, février 1984 page C2-463
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 CALCIUM IN AN O S M I U M - T R E A T E D B I O L O G I C A L SPECIMEN
S. Blaineau, J. Amsellem and G. Nicaise
Laboratoire d'Histologie et Biologie Tissulaire, Universite Claude Bernard, 69622 Villeurbanne, France
Résumé - Le nombre atomique moyen du tissu cardiaque post-fixe au tétroxyde d'osmium est 7 - 10, ce qui permet la microanalyse X du Ca par la méthode du conti- nuum. En spectromëtrie à dispersion de longueur d'onde, la concentration apparente du Ca est augmentée de 1,5 fois par la présence de l'osmium.
Abstract - The average atomic number of heart tissue post-fixed by osmium tetroxide is Z - 10. Thus this permits quantitative X-ray microanalysis of Ca by the continuum method. Employing this method, the apparent concentration of Ca measu- red with wavelength dispersive spectrometry is increased 1.5 times by 0s.
Although it is generally recommended to avoid heavy metal staining when performing quantitative X-ray microanalysis (e.g.l).it is tempting to use at least osmium to improve the natural contrast of the tissue .
For absolute quantitation with the continuum method, the bias introduced by the use of 0s (attenuation of specific signals) can be corrected, provided that Z
z/A is known and that Z< 20 (2,3). To estimate these values, we used frog heart muscle post-fixed with various concentrations (0%, 1%, 2%) of 0s04 in the fixative for variable length of time (lh, 2h, 4 h ) . The tissue was then dehydrated and embedded in epon, and its 0s content was analyzed by destructive chemical analysis at the
"Service Central d'Analyse du CNRS" Vernaison, France. The results varied little with time or concentration ; for a concentration of 2% of the fixative during lh, the 0s content of the tissue was very close to 4%, which gives a Z -10 and a Z^'/A = 5.21 ; for non osmium treated tissue Z" /A = 3.9, a value somewhat superior to that predicted by Hall et al (4).
Harmaline, a hallucinogenic drug, is known to induce an increase of intracellular free Ca in frog heart, inducing a contracture. This Ca can come from the extra- cellular milieu or from intracellular reservoirs (5). Pieces of frog heart were incubated 10 mn in either :
- control Ringer
+ +- a harmaline 3,5 mM Ringer (which increases the Ca concentration^
- or harmaline Ringer where Li is substituted for Na (the Ca / Na exchange mechanism was blocked)
The heart tissue was then fixed in glutaraldehyde and post-fixed in 0s04. After epon embedding, sections (150 nm thick) were collected on aluminium grids and ana- lyzed in a CAMEBAX-TEM microscope equipped with an oblique crystal spectrometer.
A Ca standard 5.25 mM was made by dissolving a Ca-macrocyclic polyether in araldite and the analysis proceeded as described in (6) ; however, in this experiment, the continuum (W) estimation was obtained by 15 successive 40 sec. counts of the back- ground at sin 6 = 0.400 for each analyzed spot.
The Ca concentration is nearly the same in myofilaments and mitochondria after incubation in Ringer ; it doubles after harmalin-Ringer, which agrees with cyto- chemical and physiological predictions. After harmaline-Li-Ringer there is still an increase of the Ca content in the myofilaments but not in the mitochondria.
Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:19842105
C2-464
JOURNAL DE PHYSIQUE
It i s l i k e l y t h a t those c o n c e n t r a t i o n changes r e f l e c t a s t i l l imprecise p h y s i o l o g i - c a l event.
I n t h i s case, t h e comparison between t h e Ca s i g n a l from t h e two types o f organelles i n v a r i o u s experimental c o n d i t i o n s i s r e l e v a n t because t h e 0s content (measured by energy d i s p e r s i v e spectroscopy) i s n o t s i g n i f i c a n t l y d i f f e r e n t , n e i t h e r i n t h e same section, nor i n d i f f e r e n t l y t r e a t e d blocks. However the absolute c o n c e n t r a t i o n (12.8 -13.7 mmol/kg of epoxy embedded t i s s u e i n Ringer c o n t r o l -uncorrected) are excessive i f compared t o t h e r e s u l t s obtained by o t h e r methods (7,8), and probably non p h y s i o l o g i c a l . The
Z'/A
c o r r e c t i o n i m p l i e s t h a t the apparent Ca c o n c e n t r a t i o n i n osmium t r e a t e d t i s s u e(Z"/A
= 5.21)is
lower than t h e Ca c o n c e n t r a t i o n w i t h o u t osmium(t"/A
= 3.9), and than t h e r e a l one. I n a d d i t i o n , t h e use o f osmium t e t r o x i d e has been shown t o d i s s o l v e t i s s u e calcium i n c e r t a i n cases ( 9 ) .Systematic measurements o f l a r g e areas o f h e a r t muscle s e c t i o n s were made w i t h os- mium-treated and non t r e a t e d t i s s u e t o estimate the e f f e c t o f 0s on t h e Ca s i g n a l . Contrary t o what expected, i n our c o n d i t i o n s (WDS a n a l y s i s ) t h e Ca s i g n a l i s two times h i g h e r i n osmium-treated (10 = 0.038
+
0.001) than i n non t r e a t e d t i s s u e ( I@ = 0.018 t 0.002), and t h e Ca concentration i s apparently 2.9 times h i g h e r i n osmium t r e a t e d t i s s u e (10). A s i m i l a r phenomenon has been described by o t h e r authors (11, 12), and can be a t t r i b u t e d t o an heavy- element-induced increase i n s c a t t e r e d e l e c t r o n s , thus e x c i t i n g calcium atoms o u t s i d e o f the analyzed spot.Thus, i f the a n a l y s i s i s made i n wavelength-dispersive X-ray spectrometry, an osmium t e t r o x i d e p o s t - f i x a t i o n induces a 1.5 times increase i n average apparent Ca concen- t r a t i o n o f the specimen. But i t i s always p o s s i b l e t h a t s i g n i f i c a n t amounts of Ca are r e d i s t r i b u t e d d u r i n g chemical f i x a t i o n and if one wants t o know p h y s i o l o g i c a l concentrations i t i s necessary t o immobilize t i s s u e Ca by quick f r e e z i n g . A f t e r t h i s step
,
0s may s t i l l be needed e i t h e r t o vapour-stain cryosections o r as an a d d i t i v e t o the s o l v e n t i n f r e e z e - s u b s t i t u t i o n . Provided t h a t i t s measured concentration i s n o t t o o h i g h and does n o t vary s i g n i f i c a n t l y over t h e d i f f e r e n t analyzed compart- ments, osmium can be used i n a q u a n t i t a t i v e study of o t h e r elements ; th e present study shows t h a t i t i s necessary t o make a p a r a l l e l a n a l y s i s on c o n t r o l t i s s u e n o t t r e a t e d w i t h osmium, as the s i g n a l f o r t h e s t u d i e d element may be enhanced r a - t h e r than attenuated by the presence o f 0s.T h i s work was c a r r i e d o u t i n t h e C e n t r e d e N i c r o s c o p i e E l e c t r o n i q u e A p p l i q u P e a l a B i o l o g i e e t 1 a G e o l o g i e ( U n i v e r s i t e C1. Bernard -Lyon I ) , w i t h t h e h e l p o f f u n d s from t h e C.N.R.S. (L.A. 0 4 0 2 4 4 ) and t h e D.G.R.S.T. (A.C.C. 78-7-0873, A.S. 79-7-0281).
( 1 ) QUINTANA C., HALPERN S. eds, Microanalyse X en B i o l o g i e , SFME, Paris,(1983).
( 2 ) HALL T.A., Plicrobeam Analysis i n Biology, LECHENE C.P., WARNER R.R. eds, Academic Press, N.Y. (1979) 185.
( 3 ) SHUIIAN H., SOMLYO A.V., SOMLYO A.P., Ultramicroscopy
1
(1976) 317.( 4 ) HALL T.A., ANDERSON A.C., APPLETON T., J. Microsc. 99 (1973) 177.
(5) BLAINEAU S., ROCHE M., ROUGIER O., J.Molec. c e l l . C z d i o l . 12 (1980) 77.
(6) NICAISE G., AMSELLEM J., BLAINEAU S., HEMMING F., I C X O M 10,'ibulouse (1983).
(7) SOMLYO A.P., SOMLYO A.V., SHUIIAN H., J . C e l l B i o l . 81, (1979) 316.
( 8 ) WENDT-GALLITELLI M.F., JACOB R., J . Molec. c e l l . ~ G d i o l . 14 (1982) 487.
(9) SUER R.J., PETERS P.D., EMMINES J.P., J. C e l l Sci.
15
( 1 T 4 ) 679.(10) (Ca)o 10
Z"/Ao
(Ca) : calcium c o n c e n t r a t i o nw = , ~ ~ ( = 2.9 o : t i s s u e t r e a t e d w i t h osmium
I$
: t i s s u e w i t h o u t osmium I : s i g n a l i n t e n s i t y expressedby P-b/W-W,.
(11) MORLEVAT J.P., ROUSSIGNOL J., J.Physics ( S c i . Instrum.)
5
(1972) 332.(12) GALLE P.,
