X-RAY MICROSCOPY AND MICROANALYSIS : INTRODUCTORY SURVEY

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X-RAY MICROSCOPY AND MICROANALYSIS : INTRODUCTORY SURVEY

V. Cosslett

To cite this version:

V. Cosslett. X-RAY MICROSCOPY AND MICROANALYSIS : INTRODUCTORY SURVEY. Jour- nal de Physique Colloques, 1984, 45 (C2), pp.C2-59-C2-63. �10.1051/jphyscol:1984215�. �jpa-00223821�

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Colloque C2, suppl6ment au n02, Tome 45, f6vrier 1984 page C2-59

X-RAY MICROSCOPY AND MICROANALYSIS : INTRODUCTORY SURVEY

V.E. Cosslett

Cavendish Laboratory, Cambridge, U.K.

Rdsum6 - On observe un renouveau dans l'utilisation des rayons X en micro- scopie dans deux cas diffdrents : a) avec un fort pouvoir de rdsolution gSo- m6trique en biologie et en science des mat6riaux ; b) sans trop s'attacher B la rCsolution g6om6trique en biologie, par rayonnement synchrotron.

La microanalyse par rayons X continue son dCveloppement 2 la fois dans la compr6hension fondamentale et dans l'instrumentation. Le choix des tdmoins reste le problsme le plus important.

Abstract - In recent years there has been a certain revival in the use of X-rays for microscopy. It has taken place in two quite different spheres: at low resolution for some practical purposes in biology and materials science and at higher resolution in mainly biological applications with the aid of synchrotron radiation.

Microanalysis with X-rays continues to develope in both fundamental under- standing and in instrumentation. The major problem now seems to be to establish reliable standards.

1. Introduction

At the Cambridge conference in 1956 (I), counted later as the first ICXOM meeting, the contributions were approximately equally divided between contact, projection and reflection microradiography, with small sections on microdiffraction and microanalysis. Through the succeeding meetings of the series the relative proport-

ions have changed considerably, so that at ICXOM 9 in The Hague projection micro- radiography was represented by a single paper only and X-ray optics not at all.

Microanalysis had become the main interest, carried out by several other methods as well as by X-rays. At this 10th meeting the balance has been restored to a sli-ght extent, mainly because of the entry of synchrotron radiation as an intense source of X-rays and its combination with the zone plate lens. A comprehensive survey of these methods of X-ray imaging has recently appeared (2).

The technique of microanalysis by means of X-ray spectrometry has become so wide- spread that it is no longer possible to covez all of its applications here. The basic physics of it, together with the details of instrumentation on the one hand and on the other of the treatment of the recorded data, have been set out in a lengthy multi-authored treatise ( 3 ) . At the same time manufacturers have made available

the necessary hardware, and to some degree the associated software as well. The same may be said of the newer method of electron energy loss spectrometry. In both technologies their use has progressed faster than the laying of sound foundations:

reliable standards have still to be devised and agreed upon. Surveys of these methods and related analytical techniques have recently been published by Werner ( 4 )

and by Cazaux (5).

2. X-ray Microscopy

Theory As a reminder of the principles of the projection technique the essentials are shown in Figure 1. A point source of X-rays behind an object casts an image by simple geometrical projection on to a surface at a distance, so that the magnific- ation is given by M=a/b. The image may be recorded photographically, by an array of detectors or by a single detector if the source is scanned. Contrast is

Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:1984215

C2-60 JOURNAL DE PHYSIQUE

d e t e r m i n e d by v a r i a t i o n o f X-ray a b s o r p t i o n i n t h e o b j e c t , s h a r p n e s s (and hence r e s o l u t i o n ) by F r e s n e l d i f f r a c t i o n and penumbra a f f e c t s . The p e r i o d o f exposure w i l l depend j o i n t l y o n t h e i n t e n s i t y of t h e r a d i a t i o n and on t h e s e n s i t i v i t y of t h e

d e t e c t i o n system.

Fig. 1 -. Formation of Fresnel fringes by diffraction of illumination from source S at straight-edge 0. Right: the variation of intensity across any subne- quent plane of observation.

The r e l a t i v e i n f l u e n c e of t h e s e f a c t o r s h a s been d i s c u s s e d by C o s s l e t t and Nixon ( 6 ) and o t h e r s . The g e n e r a l c o n c l u s i o n was t h a t p r o j e c t i o n X-ray microscopy p r o v i d e s a u s e f u l e x t e n s i o n of o p t i c a l methods f o r specimens t h a t a r e o p t i c a l l y opaque o r t o o t h i c k f o r l i g h t microscopy. It i s l i m i t e d by t h e i n t e n s i t y of a t t a i n a b l e s o u r c e s of X - r a d i a t i o n . T h i s i n t e n s i t y i n t u r n i s l i m i t e d p r i m a r i l y by t h e p r o p e r t i e s of t h e t a r g e t m a t e r i a l : t h e i n c i d e n t e l e c t r o n beam may b e of v e r y h i g h i n t e n s i t y , b u t t h e e l e c t r o n s n o t o n l y d e l i v e r h e a t t o t h e t a r g e t b u t a l s o a r e s c a t t e r e d i n i t . It i s n e c e s s a r y t o u s e a t h i n t a r g e t i n o r d e r t o o h t a i n a w e l l d e f i n e d s m a l l X-ray s o u r c e . Heat d i s s i p a t i o n t h e n becomes t h e main l i m i t i n g f a c t o r i n t h e t a r g e t and hence on e x p o s u r e time. Some improvement c a n b e o b t a i n e d by s c a n n i n g t h e e l e c t r o n probe and some by t h e u s e of a n image i n t e n s i f i e r ( 7 ) . The i n t e n s i t y of t h e e l e c t r o n beam c a n o n l y be r e d u c e d , however, t o r h e p o i n t where quantum s t a t i s t i c s i n t h e image s e t s a l i m i t of i t s own, by t h e s i g n a l / n o i s e r a t i o .

I n s t r u m e n t a t i o n

The t y p e of X-ray p r o j e c t i o n microscope i n t r o d u c e d by C o s s l e t t and Nixon ( 6 ) h a s c o n t i n u e d t o be u s e d , b u t t h e r e h a s been a n i n c r e a s i n g tendency f o r t h e t e c h n i q u e t o b e a d a p t e d t o a s c a n n i n g EM. For moderate r e s o l u t i o n s e v e r a l models a r e

manufactured by t h e Wardray Company a t Oxford, i n c l u d i n g one f o r 100-200kV. E e t a i l s of t h e s e i n s t r u m e n t s and v a r i c u s o t h e r s made i n i n d i v i d u a l l a b o r a t o r i e s a r e des- c r i b e d by Ely ( 8 ) . The u s e o f a n SEM f o r X-ray r a d i o g r a p h y h a s been s e t o u t by a number of a u t h o r s , Brfinger ( 9 ) , Cazaux e t a 1 ( l o ) , Horn and W a l t i n g e r (1 1) amongst o t h e r s . The e x p e r i m e n t a l a p p a r a t u s employed i s t o some e x t e n t t r e a t e d i n t h e wider s u r v e y of i n s t r u m e n t a t i o n i n e l e c t r o n o p t i c s and X-ray s p e c t r o s c o p y by Mulvey jn t h i s Congress ( 1 2 ) .

The u s e of s y n c h r o t r o n r a d i a t i o n f o r X-ray microscopy ( F i g u r e 2) h a s been e x p l o r e d i n a number of l a b o r a t o r i e s i n t h e United S t a t e s and i n Europe. The p o l y c h r o m a t i c beam may be d i s p e r s e d by means o f a g r a t i n g a t g r a z i n g i n c i d e n c e , and c o n c e n t r a t e d f o r imaging by one o r more zone p l a t e s . S u i t a b l e a r r a n g e m e n t s h a v e , b e e n d e s c r i b e d by Schmahl e t a 1 ( 1 3 ) , S i l k ( 1 4 ) , K i r z e t a 1 ( 1 5 ) , S a y r e (16) and o t h e r s . See a l s o c o n t r i b u t i o n s t o t h i s Congress by H a s t i n g s and Howells ( 1 7 ) , Schmahl e t a 1 (181, Kozlenkov and S h u l g i n (19) and Burge e t a 1 ( 2 0 ) .

A p p l i c a t i o n s X-ray microscopy i s r e s t r i c t e d t o a r e s o l v i n g power of t h e same o r d e r a s t h a t of t h e l i g h t microscope, 0.2-0.5pm. W i t h i n t h e s e l i m i t a t i o n s , however, i t h a s found a range of a p p l i c a t i o n s , m o s t l y a t a p o o r e r r e s o l u t i o n where many of t h e f a c t o r s mentioned above do n o t i n t e r f e r e w i t h performance.

Some of t h e a p p l i c a t i o n s i n m a t e r i a l s s c i e n c e and i n b i o l o g y a r e d e s c r i b e d i n

"Microfocal Radiography" ( 8 ) . The r a n g e of t h i s X-ray t e c h n i q u e i s d e f i n e d by Sharpe and P a r i s h (21) a s t h a t f o r which t h e s o u r c e s i z e l i e s between 100um and

" t h e wavelength of X-rays" (0.lpm). They d e s c r i b e t h e t e c h n o l o g y i n v o l v e d and a number of a p p l i c a t i o n s i n e n g i n e e r i n g and r e a c t o r p h y s i c s : t u r b i n e b l a d e s , i n t e g - r a t e d c i r c u i t s , carbon f i b r e s , aluminium a l l o y s . Some m e t a l l u r g i c a l a p p l i c a t i o n s of t h e s c a n n i n g t e c h n i q u e s a t a beam v o l t a g e of 30kV a r e mentioned by Cazaux e t a 1 ( 1 0 ) .

I n b i o l o g y and medicine i t i s u s u a l l y d e s i r a b l e t o u s e o a lower v o l t a g e , e x c i t i n g s o f t e r X-rays. The wavelength r a n g e of I-lOnm (10-100A) i s p r e f e r a b l e , correspond- i n g t o a v o l t a g e of21-O.IkV, a l t h o u g h a much h i g h e r v o l t a g e i s needed t o produce a n X-ray beam of a d e q u a t e i n t e n s i t y . B i o l o g i c a l a p p l i c a t i o n s i n t h i s r e g i o n have been d e s c r i b e d by Schmahl e t a 1 (131, who imaged diatom and b r a i n c e l l s of t h e r a t . F u r t h e r i n v e s t i g a t i o n s w i t h s y n c h r o t r o n r a d i a t i o n a r e d e s c r i b e d by t h e same a u t h o r s i n a f o l l o w i n g paper i n t h i s s e s s i o n of t h e c o n g r e s s ( a b s t r a c t p . 2 3 ) . For t h i c k e r b i o l o g i c a l specimens, and e s p e c i a l l y f o r h a r d t i s s u e s , more p e n e t r a t i n g X-rays and hence a h i g h e r v o l t a g e a r e r e q u i r e d . Using a p p a r a t u s o p e r a t i n g i n t h e r a n g e 1-30kV Saunders (22) h a s s t u d i e d t h e microangiography of t h e nervous system, p a r t i c u l a r l y t h e human c e r e r b a l c o r t e x , a f t e r i n j e c t i o n of c o n t r a s t m a t e r i a l . For examining h i s t o l o g i ~ a l s e c t i o n s he h a s c o n s t r u c t e d a low v o l t a g e X-ray t u b e f o r wavelengths of 2-12A (Saunders ( 2 3 ) ) . For t h e p u r p o s e s of X-ray microtomography Brunger ( 9 ) u s e s a beam a t 30kV.

A comprehensive survey of t h e b i o l o g i c a l u s e of t h e p r o j e c t i o n X-ray microscope, a t beam v o i t a g e s up t o 80kV, h a s been g i v e n by Buekland-Wright ( 2 4 ) . He d e a l s w i t h t h e r e l e v a n t t e c h n i q u e s , i n c l u d i n g specimen p r e p a r a t i o n and image r e c o r d i n g , a s w e l l a s t h e i r a p p l i c a t i o n i n t h e s t u d y of b o t h h a r d and s o f t t i s s u e s . For t h e former t y p e of specimen a h i g h e r v o l t a g e a s w e l l a s s p e c i a l p r e p a r a t i v e methods a r e needed. These a r e d i s c u s s e d by Fearnhead and Knight (25) i n c o n n e c t i o n w i t h t h e m i c r o r a d i o g r a p h y of m i n e r a l i z e d t i s s u e s , b o t h t o o t h and bone. Other a p p l i c a t i o n s t o a wide v a r i e t y of b i o l o g i c a l m a t e r i a l , b o t a n i c a l a s w e l l a s z o o l o g i c a l , a r e d e s c r i b e d by Van Emden and Ely ( 2 6 ) . They a l s o d i s c u s s t h e e f f e c t of low d o s e s of X-rays on l i v i n g organisms, which a r e sometimes r a d i o g r a p h e d i n t h a t c o n d i t i o n i n a i r .

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F u t u r e P r o s p e c t s

The p o s s i b i l i t i e s f o r f u r t h e r development of t h e X-ray microscope have been d i s c u s s e d i n some d e t a i l by S t u a r t (27) i n a l o n g s u r v e y w i t h many r e f e r e n c e s . He s e t s o u t t h e r e l a t i v e a d v a n t a g e s o f " t h e 19 most s i g n i f i c a n t o p t i o n s r ' i n f o u r t a b l e s . He c o n c l u d e s t h a t " t h e e l e c t r o n bombarded s o l i d / f o i l t a r g e t s o u r c e i s a n obvious p r e f e r e n c e u n l e s s t h e r e i s a compelling r e q u i r e m e n t , such a s a v e r y h i g h r a d i a n c e o r v e r y s h o r t d u r a t i o n p u l s e d o p e r a t i o n , which d i c t a t e s otherwise". The d i s - advantage of a s y n c h r o t r o n s o u r c e i s t h e need t o r a k e a n experiment t o a c e n t r a l i n s t a l l a t i o n , though i t h a s h i g h r a d i a n c e even a f t e r monochromatization. The plasma t y p e of X-ray s o u r c e i s " r a t h e r c o s t l y " , b u t i s "well s u i t e d t o micro- r a d i o g r a p h y of moving o b j e c t s " . Be d e s c r i b e s a l s o a n u n c o n v e n t i o n a l s i n g l e p o l e m i c r o f o c u s t u b e due t o Pugh and West (28) i n which t h e magnetic l e n s (of t h e Mulvey t y p e ) i s combined w i t h a s t a n d a r d t r i o d e e l e c t r o n gun; it g i v e s a l a r g e o u t p u t (up t o 50uA) a t a v o l t a g e of 1-25kV. S t u a r t ends by s t r e s s i n g t h e growing importance of a n image p r o c e s s i n g system f o r i n t e r p r e t i n g t h e image.

3 . X-ray M i c r o a n a l y s i s

The t e c h n i q u e o f u s i n g X-rays f o r l o c a l i s e d a n a l y s i s was p i o n e e r e d by C a s t a i n g (29) and developed by Duncumb ( 3 0 ) . It i s e s s e n t i a l l y a m a t t e r of m i c r o s p e c t r o m e t r y , r e l y i n g on t h e v a r i a t i o n i n c h a r a c t e r i s t i c l i n e e m i s s i o n t h r o u g h t h e p e r i o d i c t a b l e . The o r i g i n a l s i m p l e i n s t r u m e n t a t i o n h a s become i n c r e a s i n g l y c o m p l i c a t e d , a s d e s c r i b e d l a t e r i n t h i s Congress by Mulvey ( 1 2 ) . It i s now p a r t o f t h e hardware b u i l t i n t o t h e modern a n a l y t i c a l e l e c t r o n microscope, a l t h o u g h some microprobe i n s t r u m e n t s d e d i c a t e d t o X-ray a n a l y s i s a r e s t i l l produced.

The t e c h n i q u e i n a l l i t s a s p e c t s , i n c l u d i n g t h e p h y s i c a l b a s i s and t h e p r o c e s s i n g of d a t a , i s d e s c r i b e d i n t h e book by Hren e t a 1 ( 3 ) , of which a new e d i t i o n i s i n t h e p r e s s . I t s u s e i n p a r t i c u l a r a r e a s of s c i e n c e f o r q u a n t i t a t i v e p u r p o s e s i n v o l v e s s t a n d a r d r e f e r e n c e samples a n d / o r a c o m p l i c a t e d system of e x p e r i m e n t a l c o r r e c t i o n s . I n m e t a l l u r g y and m a t e r i a l s s c i e n c e t h i s i s sometimes a s t r a i g h t f o r w a r d m a t t e r , b u t can become d i f f i c u l t when t h e sample i s a t h i n f o i l ( ~ o s s i b l y of v a r y i n g t h i c k n e s s ) r a t h e r t h a n a b u l k specimen. The problem h a s been d i s c u s s e d i n some d e t a i l by G o l d s t e i n and by Zaluzec i n c h a p t e r s 3 and 4 of r e f e r e n c e 3 . The l a t t e r s e t s o u t a p r o c e d u r e f o r s t a n d a r d l e s s a n a l y s i s i n b o t h t h i n f i l m and b u l k samples. There i s c o n t i n u i n g d i s c u s s i o n of t h e a p p l i c a b i l i t y and a c c u r a c y of c o r r e c t i o n methods i n o t h e r s e s s i o n s of t h i s Congress.

I n b i o l o g y t h e s i t u a t i o n i s somewhat d i f f e r e n t because of t h e n a t u r e o f t i s s u e s , s o t h a t sample p r e p a r a t i o n , specimen t h i c k n e s s and mode of mounting must be t a k e n i n t o a c c o u n t . These problems a r e d e s c r i b e d i n c h a p t e r 5 o f r e f e r e n c e 3 by H a l l and Gupta, and i n many o t h e r p u b l i c a t i o n s . A v e r y u s e f u l g u i d e t o t h e complex- i t i e s of t h e a p p l i c a t i o n of X-ray m i c r o a n a l y s i s i n b i o l o g y i s t h e c o m p i l a t i o n of p a p e r s from t h e a n n u a l SEM m e e t i n g s ( 3 1 ) . One s e c t i o n of i t s e t s o u t t h e v a r i o u s s t e p s i n q u a n t i t a t i v e s t u d i e s : "spectrum h a n d l i n g and background removal p r o c e d u r e s , s t a n d a r d s , t h e o r y of q u a n t i t a t i o n problems and s o u r c e s of e r r o r " . A p a r t i c u l a r l y v a l u a b l e paper i s t h a t by Chandler ( 3 2 ) , which d i s t i n g u i s h e s between t h i c k , t h i n and u l t r a t h i n samples, t h a t r e q u i r e d i f f e r e n t t r e a t m e n t s f o r q u a n t i t a t i o n . The q u e s t i o n of t h e u s e o f s t a n d a r d s i n b i o l o g i c a l microprobe a n a l y s i s i s s t i l l v e r y much a s u b j e c t of c o n t e n t i o n , b u t a t a p o o r e r l e v e l of e r r o r ('1.5-10%) t h a n i n m e t a l s . It h a s been d i s c u s s e d by Roomans (33) and by Russ (34) a s w e l l a s by t h e a u t h o r s mentioned above. The r e c e n t work by Hagler e t a 1 (35) seems t o promise a d e f i n i t i v e answer t o t h e problem. The e f f e c t o f t h e e l e c t r o n beam on t h e specimen i s a s e p a r a t e m a t t e r , however, which i s d i s c u s s e d i n many of t h e s e p a p e r s ; i t may be minimised by c r y o - t e c h n i q u e s , b u t n o t c o m p l e t e l y a v o i d e d .

Conclusion

It i s tempting t o make a comparison of t h e X-ray probe t e c h n i q u e w i t h o t h e r micro- a n a l y t i c a l methods. T h i s i s p o i n t l e s s i n any r e a l i s t i c s e n s e , however, b e c a u s e t h e y d i f f e r w i d e l y i n t h e t y p e of a p p l i c a t i o n f o r which e a c h i s b e s t s u i t e d . Energy l o s s

spectrometry may be optimal for detecting very small aggregations of a single element, but X-ray microanalysis is likely to be the only means of elemental analysis of a multi~om~onent specimen. In a broader view the principles and limits of accuracy of the various methods have been discussed and tabulated by both Cazaux

(5) and by Werner (4). The extent to which some technique is applicable to a particular problem can often be judged in meetings such as the present occasion.

References

(1) Cosslett, V.E. ~ngstrgm, A. and Pattee, H.H. (Eds.) X-ray Microscopy and and Microradiography. Academic Press, New York, 1957.

(2) Ultrasoft X-ray Microscopy: its Application to Biological and Physical Sciences (ed. D.R. Parsons). New York Academy of Sciences (1980).

(3j Hren, J.J., Goldstein, J.I. and Joy, D.C. Introduction to Analytical Electron Microscopy. Plenum Press, New York, 1979.

(4) Werner, H.W. in Electron Microscopy 1980, Vo1.3 (eds. P. Brederoo and V.E. Cosslett), p.200.

(5) Cazaux, J., J. Microsc. Spectrosc. Electron.,

L

(6) Cosslett, V.E. and Nixon, W.C. X-ray Microscopy. University Press, Cambridge, 1960.

(7) Anderton, H. and Smith, K.C.A. In Fourth International Congress on X-ray Optics and Microanalysis (ed. R. Castaing, P. Deschamps and J. Philibert).

Hermann, Paris; p.426, 1965.

(8) Ely, R.V. (ed.) Microfocal Radiography. Academic Press, London, 1980.

(9) Brunger, W. Proc. 9th Internat. Cong. Elect. Microsc., Toronto

I

53

(11) Horn, H.R.F. and Waltinger, H.G. in Electron Microscopy 1980, Vol.1.

(eds. P. Brederoo and G. Boom) p. 94.

(12) Mulvey, T. in the vdlume of abstracts, p.40.

(13) Schmahl, G., Rudolph, B., Niemann, B. and Christ, 0. in Ultrasoft X-ray Microscopy. (ed. D.F. Parsons). New York Acad. Sci.,

342

(15) Kirz, J., Burg, R. and Rarback, H. ibid. p. 135.

(16) Sayre, D. ibid. p. 387.

(17) Hastings, J.B. and Howells, M.R. The volume of abstracts p.21.

(18) Schmahl, G., Rudolph, D. and Niemann, B. The volume of abstracts p.23 (19) Kozlenkov, A.1. and Shulgin, A.I. The volume of abstracts, p.42

(20) Burge, R.E., Duke, P.J., Macdowell, A., Michette, A., Miller, A., Rosser, R., Simpson, M. and West, J.B. The volume of abstracts p.25.

(21) Sharpe, R.S. and Parish, R.W. in ref. 8, chapter 2.

(22) Saunders, R.L. de C.H. in ref. 8 chapter 3.

(23) Saunders, R.L. de C.H. in "The Encyclopaedia of Microscopy and Microtechnique".

(ed. P. Gray). Van Nostrand Reinhold, New York. 1973, p.603 (24) Buckland-Wright, J.C., in ref. 8, chapter 4.

(25) Fearnhead, R.W. and Knight, D. in ref. 8,chapter 5.

(26) Van Emden, H.F. and Ely, R.V., in ref. 8, chapter 6.

(27) Stuart, P.R., in ref. 8, chapter 7.

(28) Pugh, D.J. and West, P.D. in Developments in Electron Microscopy and Analysis 1977, (ed. D.L. Misell). Institute of Physics, Bristol and London, p.29.

(29) Castaing, R. Thesis, Paris 1951 (30) Duncumb, P. Thesis, Cambridge, 1957.

(31) Basic Methods ,in Biological X-ray Microanalysis (ed. G.M. Roomans and J.D. Shelbourne). Scanning Electron Microscopy Inc., AMF OIHare, 1983.

(32) Chandler, J. ref. 3 1 p. 1.

(33) Roomans, G.M. ref.31 , p.251.

(34) Russ, J. ref. 31, p.261

(35) Hagler, K.H., Lopez, L.E., Flores, J.S., Lundswick, R.J. and Buja, M.L.

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