QUANTITATIVE MICROANALYSIS OF CALCIUM SUBCELLULAR COMPARTMENTS IN RESIN-EMBEDDED TISSUE SECTIONS (X-RAY WAVELENGTH DISPERSIVE SPECTROMETRY)

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QUANTITATIVE MICROANALYSIS OF CALCIUM SUBCELLULAR COMPARTMENTS IN

RESIN-EMBEDDED TISSUE SECTIONS (X-RAY WAVELENGTH DISPERSIVE SPECTROMETRY)

G. Nicaise, J. Amsellem, S. Blaineau, F. Hemming

To cite this version:

G. Nicaise, J. Amsellem, S. Blaineau, F. Hemming. QUANTITATIVE MICROANALYSIS OF CAL-

CIUM SUBCELLULAR COMPARTMENTS IN RESIN-EMBEDDED TISSUE SECTIONS (X-RAY

WAVELENGTH DISPERSIVE SPECTROMETRY). Journal de Physique Colloques, 1984, 45 (C2),

pp.C2-461-C2-462. �10.1051/jphyscol:19842104�. �jpa-00223771�

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JOURNAL DE PHYSIQUE

Colloque C2, supplément au n°2, Tome 45, février 1984 page C2-461

QUANTITATIVE MICROANALYSIS OF CALCIUM SUBCELLULAR COMPARTMENTS IN RESIN-EMBEDDED TISSUE SECTIONS (x~RAY WAVELENGTH DISPERSIVE SPECTROMETRY)

G. Nicaise, J . Amsellem, S. Blaineau and F. Hemming

Laboratoire d'Uistologie et Biologie Tissulaire, Universite Claude Bernard, 69622 Villeurbanne, France

Résumé - Une méthode de microanalyse quantitative est mise au point et utili- sée pour déterminer le contenu cal ci que de granules intracellulaires d'un diamètre moyen de 0,4 ym. L'analyse est faite en spectrométrie à dispersion de longueur d'on- de après congélation rapide, cryosubstitution et inclusion dans l'épon.

A b s t r a c t - A method o f q u a n t i t a t i v e microanalysis i s described and used t o determine the calcium content o f i n t r a c e l l u l a r g r a n u l e s , 0.4 ym i n average diameter.

The analysis i s made by wavelength d i s p e r s i v e spectrometry a f t e r q u i c k - f r e e z i n g , f r e e z e - s u b s t i t u t i o n and embedding i n epon.

I - SPECIMEN PREPARATION

A good localization ofdiffusible elements can be obtained by analytical electron microscopy of unfixed frozen tissue sections /1,2/, but cryosections are difficult to prepare and give a relatively poor image. There is increasing evidence that it is possible to preserve the physiological distribution of important elements like calcium (Ca) through freeze substitution and embedding in resins /3,4,5,6/.

In the present study, pieces of Mytiius retractor muscle were shock-frozen by slam- ming on a helium-cooled copper block /4,7/ or by dipping into freon 22 cooled by liquid nitrogen, then substituted in ethanol, acetone or tetrahydrofuran (3 days at - 85°C) in presence of 10 mM oxalic acid and calcium free molecular sieve (3A PR0LA- B0); after progressive warming, the tissues were embedded in epon. Sections (=150 nm thick) were floated onto either distilled water or glycerol (dehydrated by molecular sieve) and collected on aluminium grids without supporting film. In our case /as in 5/ the ahydrous sectioning recommended by Ornberg and Reese / 4 / was probably not needed. Control blocks were made with conventional glutaraldehyde fixation, followed by dehydration and embedding in epon.

II - STANDARDS

For epoxy embedded tissues, an indirect peripheral standard can be made by adding a sulphur-containing molecule to the resin HI ; we used epon with Thiokol LP3 (CIBA- GEIGY), and the final sulphur (S) concentration (Cs) of the standard (= 5 %) was controlled by destructive chemical analysis (in the "Service Central d'Analyse du CNRS de Vernaison").' The relative efficiency, k, of the analyser for Ca and S was measured with CaS04 crystallized on a formwar coated grid /see 8/.In our conditions k = Ica-As/I,s-ACa = 1.04 (I± specific X-ray intensity, A = atomic weight), Ca being analyzed with a crystal spectrometer (WDS) and S with an energy dispersive detector (EDS). For each analyzed spot, the Ca concentration ,n , , /,, ., N

_ Cs (P-b)c a/(W-Wo) CC a x - — — (P, b, W and WQ are defined next p.) k ( P - b )s/ ( W - W o ) Some data were also obtained with a Ca standard made by mixing a macrocyclic poly- ether complex with araldite /see 8/ ; but the Ca concentration of this standard was at best 0.12 %, and more usually 0.02 %, which gives imprecise measurements. We did not try the calcium naphtenate standard / 9 / , which could be better/see also 14/.

Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:19842104

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C2-462 JOURNAL DE PHYSIQUE

I 1 1

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ANALYSIS

We used a CAFIEBAX-TEM a n a l y t i c a l e l e c t r o n microscope, equipped w i t h an o b l i q u e c r y s - t a l spectrometer and an energy d i s p e r s i v e d e t e c t o r TRACOR TN 2000. The a c c e l e r a t i n g v o l t a g e was s e t a t 40 o r 45 kV, and t h e probe c u r r e n t ( r e g u l a t e d ) a t a 90 nA. F o r each analyzed spot, t h e s i g n a l was expressed by I = (P-b)/(W-W,) (peak minus background o v e r t h e w h i t e count o f t h e s e c t i o n minus t h e w h i t e count i n a nearby h o l e o f t h e g r i d /see

l o / .

The Ca Ka peak was measured w i t h a PET c r y s t a l a t s i n 8 = 0.3838, and t h e background a t s i n 8 = 0.400 d u r i n g 40 sec each, f o r each analyzed s p o t ; t h e w h i t e count, W, was t h e i n t e g r a l o f t h e energy d i s p e r s i v e spectrum between 4.5 and 6.0 keV. I f t h e l i q u i d n i t r o g e n t r a p i s used, t h e v a l u e o f W i s v e r y s t a b l e f o r a g i v e n area, b u t i t i n c r e a s e s by 4 % e v e r y 100 sec i f l i q u i d n i t r o g e n i s o m i t t e d . No mass l o s s was d e t e c t e d i n t h e t i m e s c a l e o f o u r analyses.

A t t e n u a t i o n ("shadowing") o r e x a g g e r a t i o n ( n o i s e ) o f t h e s i g n a l were found when ap- p r o a c h i n g t h e g r i d bars, even when t h e s e c t i o n was on t h e s u p e r i o r s i d e o f t h e g r i d ; shadowing and n o i s e were a l s o found toward t h e g r i d p e r i p h e r y , w i t h b o t h EDS and WDS /see

l o / .

P a r t i a l improvement c o u l d be o b t a i n e d w i t h an o r i g i n a l g r a p h i t e - a l u m i n i u m g r i d h o l d e r . As a r u l e , t h e measurements were o n l y t a k e n on t h e 16 most c e n t r a l ho- l e s o f t h e 200 mesh aluminium g r i d , and more t h a n 10 um away f r o m t h e g r i d bars.

I n these c o n d i t i o n s , we can q u a n t i f y 5 mM Ca p e r kg o f epon /11/, w i t h good s a f e t y (P = 2b), w i t h o u t making assumptions on peak d e c o n v o l u t i o n s .

I V

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RESULTS

The a n t e r i o r byssus r e t r a c t o r muscle o f M y t i l u s i s a v e r y s i m p l e and w e l l ordered t i s s u e , and t h e c e l l components a r e r e l a t i v e l y easy t o i d e n t i f y i n u n s t a i n e d l o n g i - t u d i n a l s e c t i o n s . The g l i o - i n t e r s t i t i a l granules,a presumed Ca s t o r e /12/ a r e o v a l , w i t h a s m a l l diameter o f 0.2 pm and a l a r g e diameter o f 0.7 vm, which i s c l o s e t o t h e s i z e of t h e beam i n o u r c o n d i t i o n s o f a n a l y s i s ( p r e f e r e n t i a l l y u s i n g a small aper- t u r e o f t h e Wehnelt). A f t e r c r y o s u b s t i t u t i o n , t h e m y o f i l a m e n t areas c o n t a i n 10 t o 20 mmol Ca/kg o f epoxy embedded t i s s u e /11/, v a l u e s v e r y c l o s e t o t h a t o b t a i n e d a f t e r g l u t a r a l d e h y d e f i x a t i o n , and which compare w e l l w i t h t h e l i t e r a t u r e d a t a o b t a i n e d b y d e s t r u c t i v e chemical a n a l y s i s /see 13/. The g l i o - i n t e r s t i t i a l g r a n u l e s g i v e s i m i l a r r e s u l t s a f t e r aldehyde f i x a t i o n , b u t w i t h t h e c r y o t e c h n i q u e s t h e i r Ca c o n t e n t i s always s u p e r i o r (up t o 180 mmol/kg) and more v a r i a b l e .

T h i s w o r k was c a r r i e d o u t i n the C e n t r e d e ~ 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 k e l a b i o l o g i e e t l a ~ d o l o g i e ( U n i v e r s i t d C l a u d e B e r n a r d

-

^{L y o n}^I),^{w i t h}the h e l p o f f u n d s f r o m t h e C.N.R.S. ( L . A . 040 244) a n d t h e D.G.R.S.T. (A.C.C. 78 7 0873, A . S . 79 7 281) 1

-

DORGE A., RICK R., GEHRING K., MASON J., THURAU K., J. M i c r . B i o l . C e l l .

22

(1975) 205.

2

-

SOMLYO A.V., SHUMAN H., SOMLYO A.P., J. C e l l B i o l

. ⁷⁴

(1977) 828.

3

-

GEYER G., HALBHUBER K.J., BENSER A., Acta histochem. 48 (1974) 257.

4

-

ORNBERG R.L., REESE T.S., Feder. Proc.

39

(1980) 280271 5

-

NEUMANN D., JANOSSY A.G.S., J. Microscopy 120 (1981) 73.

6

-

HARVEY D.M.R., J . Microscopy 127 (1982) 2 0 r

7

-

JESSEN H., PETERS P.D., HALL

m.,

J . C e l l S c i .

3

(1974) 359.

8

-

CHANDLER J.A., J . Microscopy 106 (1976) 291.

9

-

ORNBERG R., REESE T., i n " M i c F r o b e A n a l y s i s o f B i o l o g i c a l Systems" (1981) Academic Press, N.Y., 213.

10- HALL T.A., ANDERSON H.C., APPLETON T., J. Microscopy 99 (1973) 177.

11- lmmol/kg o f epoxy embedded t i s s u e = 5.3 mmol/kg d r y w z g h t , 1.2 mmol/kg of wet t i s s u e and 1.5 mmol/l o f c e l l H20 (assuming 78 % o f w a t e r i n t h e t i s s u e ) . 12- HEMMING F.J., NICAISE G., B r a i n Res.

245

(1982) 127.

13- RUEGG J.C., P h y s i o l . Rev.

2

(1971) 201.

14- DE BRUIJN W.C., i n "Scanning e l e c t r o n microscopy" (1981)

11,