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RECENT RESULTS IN EELS ELEMENTAL MAPPING OF THIN BIOLOGICAL SECTIONS
C. Jeanguillaume, J. Berry, C. Colliex, P. Galle, M. Tence, P. Trebbia
To cite this version:
C. Jeanguillaume, J. Berry, C. Colliex, P. Galle, M. Tence, et al.. RECENT RESULTS IN EELS
ELEMENTAL MAPPING OF THIN BIOLOGICAL SECTIONS. Journal de Physique Colloques,
1984, 45 (C2), pp.C2-577-C2-580. �10.1051/jphyscol:19842132�. �jpa-00223802�
JOURNAL DE PHYSIQUE
Colloque C2, supplément au n°2, Tome 45, février 1984 page C2-577
R E C E N T R E S U L T S IN E E L S E L E M E N T A L M A P P I N G OF T H I N B I O L O G I C A L S E C T I O N S
C. Jeanguillaume , J.P. Berry*, C. Colliex , P. Galle*, M. Tence and P. Trebbia"1"
Laboratoire de Physique des Solides*, Bâtiment 510, Université Paris Sud, 91405 Orsay, France
^Laboratoire de Biophysique, Faculté de Médecine, 94010 Créteil et INSERM SC 27,.France
Résumé •<• La cartographie chimique des principaux éléments contenus dans une section biologique est obtenue par traitement numérique de plusieurs images filtrées en énergie, acquises digitalement en STEM.
Abstract - Chemical mapping of the main elements within a biological section is achieved by numerical processing of several energy loss filtered images, digitally recorded with a STEM.
I - PRINCIPLE OF THE METHOD.
In electron microscopy, elemental distribution mapping, that is a two dimensional image of the composition of a specimen, is achieved in two ways : X ray mapping and Electron Energy Loss (EELS) mapping - see for instance Somlyo et al./1/for a recent review of biological applications -. The second method offers several im- portant advantages: improved spatial resolution down to the probe size in STEM (that is = 2nm), capability of providing chemical maps for low Z elements of great importance in biological sections (nitrogen, oxygen, fluorine, phosphorus...) and higher sensitivity associated to more intense cross sections for the edge of inte- rest and better detection efficiency. In the case of biological sections, this aspect is very important because one has to compare the dose required for obtaining a given signal to noise ratio on the characteristic signal, with the radiation induced damage. EELS mapping using one or several energy filtered images is there-
fore developing very quickly. High resolution microanalysis of biological specimens by electron spectroscopic imaging has been carried out either with a CTEM equipped with a Castaing Henry filter / 2 / or with a STEM fitted with a magnetic spectro- meter /3/ and / 4 / .
The main problem is due to the fact that the characteristic signal is rather small superimposed on a large background - figure 1 ^. Consequently the images have to be processed to ensure the extraction of the true chemical information. Following an idea proposed by Jeanguillaume et al./5/, several energy filtered images have to be acquired, so that for each image pixel it is possible to estimate the back- ground model curve and to extrapolate it below the signal. The smoothly varying background intensity is generally fitted with a power law curve:
B = A.AE depending on two parameters, A and R, which can be de- termined when two channels at least below the edge are recorded. The signal itself is
S = I - B , in the channel after the edge and the standard deviation 2 2 2 2 2
os can be obtained as : a<. = aj + 0n where a, = I and a„ is a function of B depending on the accuracy of the determination of R.
Associe au CNRS
Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:19842132
JOURNAL DE PHYSIQUE
.2-3 e d g e
Fig.1
-
Typical example o f EEL spectrum f o r t h e d e t e c t i o n o f a c h a r a c t e r i s t i c edge i n a b i o l o g i c a l s e c t i o n o f about 80 nm. I n t h e present case, the s i g n a l f o r the i r o nedge i s S=16031 and t h e starldard d e v i a t i o n i s o =462. F i t t i n g w i t h a law A.AE-R, :$-3achieved over a 100 eV window b e f o r e t h e edge.
his
f i g u r e a l s o p o i n t s o u t the ave- rage s i z e and p o s i t i o n o f t h e t h r e e energy windows ( w i t h r e s p e c t i v e l y 11,12 and I3 counts) g e n e r a l l y used f o r t h e e v a l u a t i o n o f S.I 1
-
MATERIALS AND METHODSThe VG microscope HB 501 equipped w i t h a Gatan spectrometer /6/7/has been i n t e r f a - ced w i t h a PDP 11/34 computer. Images a r e recorded s e q u e n t i a l l y f o l l o w i n g a d i g i t a l - l y d r i v e n r a s t e r o f 128x128 ( o r 256x256) p i x e l s . The d i f f e r e n t s i n g l e energy l o s s images a r e acquired such as t h e energy loss, determined by t h e s e l e c t i o n s l i t , i s
incremented a f t e r each l i n e : one l i n e i s t h e r e f o r e scanned n times successively, n being t h e number o f recorded energy windows ( a t l e a s t n = 3, b u t n = 4 f o r the micrographs shown i n t h i s paper). The computer records simultaneously t h i s s e t o f energy f i l t e r e d images v i a t h e b r i g h t f i e l d d e t e c t o r l o c a t e d a f t e r the spectrometer, and the dark f i e l d image v i a t h e annular d e t e c t o r b e f o r e t h e spectrometer. A l l data h a n d l i n g processes a r e c a r r i e d on a p o s t e r i o r i .
The specimen i s a biopsy o f human pulmonary t i s s u e c o n t a i n i n g several asbestos and f e r r u g i n o u s bodies. I t i s now w e l l known / 8 / t h a t some o f these s i l i c a t e p a r t i c l e s i n t h e a l v e o l a r macrophages a r e covered i n v i v o by a c o a t i n g o f i r o n hydroxide.
The sample has been prepared f o l l o w i n g t h e standard techniques: f i x a t i o n i n g l u t a - raldehyde, dehydration i n a l c o h o l , embedding i n Epon and s e c t i o n i n g w i t h an LKB ultramicrototne w t t h o u t s t a i n i n g . The average thickness i s i n the range 50 t o 70 nm.
I 1 1
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RESULTSThe p o s s i b i l i t y o f d i s p l a y i n g c h a r a c t e r i s t i c images o f low Z elements such as carbon, n i t r o g e n and oxygen has already been i l l u s t r a t e d i n /4/. The present micrographs concern d i f f e r e n t asbestos bodies o f small s i z e ( w e l l below one micron). I n the annular dark f i e l d images ( f i g . 2 a and 3a) one c l e a r l y d i s t i n g u i s h e s denser o b j e c t s appearing i n w h i t e
-
t h e a x i a l f i b e r s-
surrounded by an i r r e g u l a r contour o f granu- les, t h e f e r r u g i n o u s sheath-
o f weaker c o n t r a s t . I t corresponds t o t h e processes developed by the organism a f t e r t h e i n h a l a t i o n o f t h e atmospheric dust.The o p e r a t i n g c o n d i t i o n s and r e s u l t s o f a c q u i s i t i o n o f t h e energy f i 1 te r e d images are summartzed as f o l l o w s :
F i g . 2b
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Carbon image (edge a t 284 eV); 128x128 p i x e l s .Dwell time p e r p i x e l : 5 ms ; energy window w i d t h : 11 eV.
AE1 = 174 eV
,
I1 = 8,
I1 ave = 3484,
I1 = 14913 - 1673,
I2 = 9866 AE2 = 222 eV,
I2 = 0,
I2 ave -- 826
,
I3 = 5685 AE3 = 276 eV,
I3 = 0,
I3 ave -AE4 = 294 eV
,
I4 = 3,
I4 ave = 1171,
I4 max = 4856 Fig. 3c-
I r o n image ( edge a t 702 eV ) ; 128x128 p i x e l s .Dwell time p e r p i x e l : 10 ms ; energy window w i d t h : 10 eV.
-
272,
I1 = 951 AE1 = 534 eV,
I1 = 2,
I1 ave -AE2 = 632 eV
,
I = 1,
I2 ave = 157,
I2 = 821 AE3 = 695 eV,
I3 = 0 '3 ave = 119,
I3 = 389- 117
,
I4 = 432AE4 = 705 eV
,
I4 = 2,
I4 ave - Fig. 3d-
Oxygen image ( edge a t 532 eV); 128x128 p i x e l s .Dwell time p e r p i x e l : 7 ms ; energy window w i d t h : 10 eV.
AE1 = 338 eV
,
I1 = 20,
I1 ave = 1115,
I1 max = 3577-
2532 AE2 = 428 eV,
I2 = 19,
I2 ave = 541,
I2 max - AE3 = 525 eV,
I3 = 17,
I3 ave = 309,
I3 = 1982 AE4 = 535 eV,
I4 = 16,
I4 = 394,
I4 ma, = 5506I t i s w o r t h w i l e t o p o i n t o u t t h a t f o u r energy windows have been used i n each case, t h r e e o f them l o c a t e d b e f o r e the edge. I t improves t h e accuracy o f t h e f i t t i n g pro- cedure between the p a r e n t a l d i s t r i b u t i o n o f experimental p o i n t s and the background model curve. The o t h e r o r i g i n a l aspect l i e s i n t h e f a c t t h a t t h e energy f i l t e r e d images on t h e oxygen K edge have been recorded f i r s t , before t h e background images, so t h a t t h e eventual mass l o s s o f oxygen i s reduced. I t c o n s t i t u t e s an improvement w i t h respect t o the r e s u l t s displayed i n /4/ i n which case t h e oxygen processed image had a poorersignal t o noise r a t i o .
I V
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DISCUSSION OF THE RESULTSThe f o l l o w i n g general comments o f b i o l o g i c a l i n t e r e s t can be made. The carbon image ( 2 b ) u n d e r l i n e s the i n o r g a n i c i n c l u s i o n ( t h e b l a c k area) and a weaker carbon con- t e n t i n t h e sheath than i n t h e embedding m a t e r i a l . The i r o n image (3 c ) p o i n t s o u t t h a t the i r r e g u l a r c o a t i n g c o n s i s t s m a i n l y o f i r o n , w h i l e t h e asbestos f i b e r i n t h e center does n o t c o n t a i n any i r o n . I n t h e oxygen image ( 3 d), some a x i a l f i b e r s appear very r i c h , i t i m p l i e s t h a t they a r e l i k e l y amphiboles; t h e r e i s a l s o a weaker con- t r i b u t i o n i n t h e surrounding f e r r u g i n o u s sheath, b u t the denser i n c l u s i o n a t t h e l o - wer p a r t o f t h e micrograph e x h i b i t s a l a c k o f oxygen. I t i s l i k e l y a carbonaceous p a r t i c l e .
About the "R image" ( f i g . 3 b )
This p i c t u r e d i s p l a y s f o r each p i x e l t h e value o f t h e R parameter between 2.5(black) and 4.0(white), obtained from a f i t t i n g o f t h e background w i t h t h e power law model on t h r e e energy l o s s channels a t 60, 87 and 95 eV, below t h e s i l i c o n L edge. I t shows t h a t t h e r e e x i s t s a s t r o n g c o r r e l a t i o n between the denser p a r t iz-qhe ADF image(white areas) and i n t h e R image(dark areas), which i s due t o mass thickness v a r i a t i o n s . As a consequence, i t seems v e r y dangerous t o assume t h e same R value f o r a l l p i x e l s i n a spectroscopic image. It confirms t h e n e c e s s i t y o f the data handling processes which have been used i n t h i s work
REFERENCES
1/ SOMLYO A.P., SHUMAN H., SOMLYO A.V., proc.loth 1nt.Cong.on E l e c t r o n Microscopy, E l e c t r o n Microscopy 1982, 1, (1982) 143-150.
C2-580 JOURNAL DE PHYSIQUE
2/ OTTENSMEYER F.P., ANDREW J.W., J . o f U l t r a s t r u c t u r e Research (1980) 72,336-348.
3/ LEAPMAN R.D., FIORI C.E., GORLEN K.E., GIBSON C.C., SWYT C.R., ProcxMSA 1983, t o be pub1 ished.
4/ JEANGUILLAUME C., TENCE M., TREBBIA P., COLLIEX C., Scannina E l e c t r o n Microscopv .
-
. 1 1 (1983) 745-756.
5/ JEANGUILLAUME C., COLLIEX C., TREBBIA P., U l t r a m i c r o s c o p y 3 (1978), 137-142.
6/ JEANGUILLAUME C., KRIVANEK O.L., COLLIEX C., 1nst.Phys.ConT.Ser.
-
61 (1981 ) 189.7/ COLLIEX C., TREBBIA P., U l t r a m i c r o s c o p y 9 (1982), 259-266.
8/ BERRY J.P., HENOC P., GALLE P., PARIENTE- R., American J o u r n a l o f Pathology
83,
(1976) 427-438.
