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Quantitative X-ray microanalysis of volutin granules in acinetobacter
Abstracts of The Netherlands Society of Electron Microscopy
r e a c t i v i t y of t h i s c a r b o n t o w a r d o x y g e n is of p r a c t i c a l i n t e r e s t , b e c a u s e d e a c t i v a t e d c a t a l y s t s are r e g e n e r a t e d b y oxidation. H e r e w e use A u g e r s p e c t r o s c o p y (AES), s e c o n d a r y ion m a s s s p e c t r o m e t r y (SIMS), a n d t e m p e r a t u r e - p r o g r a m m e d reaction spectroscopy (TPRS) to s h o w t h a t o x i d a t i o n p r o v i d e s a s e n s i t i v e t e s t for the r e a c t i v i t y of c a r b o n on n o b l e m e t a l s . C a r b o n d e p o s i t s , p r e p a r e d by e x p o s i n g the m e t a l s to e t h y l e n e at d i f f e r e n t temperatures, were characterized with AES and SIMS. T h e t e m p e r a t u r e at w h i c h the carbon reacts with oxygen depends critic a l l y on the h y d r o g e n c o n t e n t of the dep o s i t a n d on the s u b s t r a t e : c a r b o n on i r i d i u m is s i g n i f i c a n t l y m o r e r e a c t i v e t o w a r d o x y g e n t h a n on p l a t i n u m . The o x i d a t i o n t e m p e r a t u r e s of c a r b o n on alumina-supported Pt a n d Ir c a t a l y s t s as d e t e r m i n e d in a c a t a l y t i c r e a c t o r are in g o o d a g r e e m e n t w i t h t h o s e m e a s u r e d on the m e t a l f o i l s in UHV.
QUANTITATIVE X-RAY MICROANALYSIS OF V O L U T I N G R A N U L E S IN A C I N E T O B A C T E R E.M. Bouw, A. M. D e i n e m a *
Boekestein
and
Technical and Physical Engineering Research Service and *Department of Microbiology, Agricultural University, Wageningen, The Netherlands
S t r a i n s of A c i n e t o b a c t e r are a b l e to a c c u m u l a t e l a r g e a m o u n t s of p o l y p h o s p h a t e w h i c h is p r e s e n t in the s o - c a l l e d v o l u t i n granules. The b a c t e r i a l c e l l s , c o n t a i n ing m a n y g r a n u l e s , w e r e c r y o s e c t i o n e d in an L K B u l t r a t o m e III c r y o - u l t r a m i c r o t o m e and s u b s e q u e n t l y t r a n s f e r r e d to a c a r b o n evaporation device. G o o d p r e s e r v e d sect i o n s c o n t a i n i n g r e c o g n i z a b l e p a r t s of the b a c t e r i a w e r e a n a l y z e d in the c o l d s t a g e of a P h i l i p s E M 4 0 0 T s c a n n i n g t r a n s m i s s i o n e l e c t r o n m i c r o s c o p e at 80 k V a c c e l e r a t i n g v o l t a g e a n d 24 ° t i l t angle. Quantification was achieved using cryos e c t i o n e d s t a n d a r d s c o m p o s e d of g e l a t i n , g l y c e r i n , K H 2 P O 4 and w a t e r . Both standard a n d s p e c ± m e n s e c t i o n s w e r e f r e e z e d r i e d p r i o r to a n a l y s i s in o r d e r to improve peak-to-background ratios. To calc u l a t e m a s s f r a c t i o n s of P in the v o l u t i n g r a n u l e s , the c o n t i n u u m - n o r m a l i s a t i o n m e t h o d of H a l l was f o l l o w e d , u s i n g an e x t r a n e o u s b a c k g r o u n d c o r r e c t i o n by m e a s u r i n g the b a c k g r o u n d i n t e n s i t y on the s u p p o r t i n g f i l m in the v i c i n i t y of the sections. M e a s u r e d v a l u e s of P in the g r a n u l e s v a r i e d f r o m 30 to 35%, w h i c h is
199
in a g r e e m e n t w i t h a n a l y s e s u s i n g o t h e r m e t h o d s a n d w h i c h s u p p o r t s the h y p o t h e s i s t h a t the v o l u t i n g r a n u l e s are a l m o s t ent i r e l y c o m p o s e d of p u r e p o l y p h o s p h a t e .
INTEGRATED IMAGE ANALYSIS AND ELECTRON E N E R G Y L O S S S P E C T R O S O C P Y IN E M W.C. de B r u i j n , C.W.J. S o r b e r , T. de J o n g and J.B. v a n D o r t * AEM Unit, Clin. Pathol. Inst. Erasmus University, PO Box 1738, Rotterdam; *Hogeschool West-Brabant, Lab Onderwijs, Etten Leur, The Netherlands
T h e c o m b i n a t i o n , Z e i s s E M 902 m i c r o s c o p e w i t h an I B A S - 2 0 0 0 i m a g e a n a l y s i s system, allows morphometrical quantitat i o n a n d i n t e g r a t i o n of i m a g e s f r o m e l e m e n t - r e l a t e d e l e c t r o n e n e r g y loss (EELS) w i t h c e l l - s t r u c t u r e - r e l a t e d inelastically scattered electron popul a t i o n s (ESI). C o n t r a s t t h r e s h o l d i n g has b e e n app l i e d to s u c h s p e c t r o s c o p i c a l i m a g e s to o b t a i n a p p r o p r i a t e a n d o b j e c t i v e segmentation. In g l u t a r a l d e h y d e - f i x e d cells, c h e m i c a l e l e m e n t s in two c e l l components have been morphologically analyzed: o n e i n t r i n s i c e l e m e n t a n d a c y t o chemical reaction product. It is s h o w n t h a t for the i n t r i n s i c n u c l e a r p h o s p h a t e and for a b a r i u m - c o n t a i n i n g r e a c t i o n p r o d u c t in the R E R ( f r o m an a r y l s u l f a t a s e r e a c t i o n ) a v o l u m e f r a c t i o n c a n be e s t i m a t e d . The use of an i o n - e x c h a n g e b e a d type of B i o - s t a n d a r d (Polaron, UK) has rec e n t l y b e e n a d v o c a t e d for e l e m e n t a l c o n c e n t r a t i o n e s t i m a t i o n s , in X - r a y m i c r o analysis. It has b e e n s h o w n t h a t a cons t a n t ratio, r e p r e s e n t i n g the r e l a t i v e concentration ( R ) of t h e s e s t a n d a r d s , c o u l d be o b t a i n e ~ . F o r e a c h t y p e of s t a n d a r d a p r o p o r t i o n a l i t y c o n s t a n t has b e e n c a l c u l a t e d , that relates R to the s t a n d a r d ' s m e a n (C) concentration value, estimated by neutron actlvatlon analysis. For quant i t a t i v e E E L S a n a l y s i s the c o n s t a n c y of R is i n v e s t i g a t e d for s t a n d a r d s c o n t a i n e ~ t h e r Ca, Co, Ni, Fe or Cu. Special a t t e n t i o n is g i v e n to d e l i b e r a t e l y i n t r o duced section thickness variations, to s h o w that, w i t h i n l i m i t s , the R v a l u e is i n d e p e n d e n t of s e c t i o n x thickness. X
.
.
r e a c t i v i t y of t h i s c a r b o n t o w a r d o x y g e n is of p r a c t i c a l i n t e r e s t , b e c a u s e d e a c t i v a t e d c a t a l y s t s are r e g e n e r a t e d b y oxidation. H e r e w e use A u g e r s p e c t r o s c o p y (AES), s e c o n d a r y ion m a s s s p e c t r o m e t r y (SIMS), a n d t e m p e r a t u r e - p r o g r a m m e d reaction spectroscopy (TPRS) to s h o w t h a t o x i d a t i o n p r o v i d e s a s e n s i t i v e t e s t for the r e a c t i v i t y of c a r b o n on n o b l e m e t a l s . C a r b o n d e p o s i t s , p r e p a r e d by e x p o s i n g the m e t a l s to e t h y l e n e at d i f f e r e n t temperatures, were characterized with AES and SIMS. T h e t e m p e r a t u r e at w h i c h the carbon reacts with oxygen depends critic a l l y on the h y d r o g e n c o n t e n t of the dep o s i t a n d on the s u b s t r a t e : c a r b o n on i r i d i u m is s i g n i f i c a n t l y m o r e r e a c t i v e t o w a r d o x y g e n t h a n on p l a t i n u m . The o x i d a t i o n t e m p e r a t u r e s of c a r b o n on alumina-supported Pt a n d Ir c a t a l y s t s as d e t e r m i n e d in a c a t a l y t i c r e a c t o r are in g o o d a g r e e m e n t w i t h t h o s e m e a s u r e d on the m e t a l f o i l s in UHV.
QUANTITATIVE X-RAY MICROANALYSIS OF V O L U T I N G R A N U L E S IN A C I N E T O B A C T E R E.M. Bouw, A. M. D e i n e m a *
Boekestein
and
Technical and Physical Engineering Research Service and *Department of Microbiology, Agricultural University, Wageningen, The Netherlands
S t r a i n s of A c i n e t o b a c t e r are a b l e to a c c u m u l a t e l a r g e a m o u n t s of p o l y p h o s p h a t e w h i c h is p r e s e n t in the s o - c a l l e d v o l u t i n granules. The b a c t e r i a l c e l l s , c o n t a i n ing m a n y g r a n u l e s , w e r e c r y o s e c t i o n e d in an L K B u l t r a t o m e III c r y o - u l t r a m i c r o t o m e and s u b s e q u e n t l y t r a n s f e r r e d to a c a r b o n evaporation device. G o o d p r e s e r v e d sect i o n s c o n t a i n i n g r e c o g n i z a b l e p a r t s of the b a c t e r i a w e r e a n a l y z e d in the c o l d s t a g e of a P h i l i p s E M 4 0 0 T s c a n n i n g t r a n s m i s s i o n e l e c t r o n m i c r o s c o p e at 80 k V a c c e l e r a t i n g v o l t a g e a n d 24 ° t i l t angle. Quantification was achieved using cryos e c t i o n e d s t a n d a r d s c o m p o s e d of g e l a t i n , g l y c e r i n , K H 2 P O 4 and w a t e r . Both standard a n d s p e c ± m e n s e c t i o n s w e r e f r e e z e d r i e d p r i o r to a n a l y s i s in o r d e r to improve peak-to-background ratios. To calc u l a t e m a s s f r a c t i o n s of P in the v o l u t i n g r a n u l e s , the c o n t i n u u m - n o r m a l i s a t i o n m e t h o d of H a l l was f o l l o w e d , u s i n g an e x t r a n e o u s b a c k g r o u n d c o r r e c t i o n by m e a s u r i n g the b a c k g r o u n d i n t e n s i t y on the s u p p o r t i n g f i l m in the v i c i n i t y of the sections. M e a s u r e d v a l u e s of P in the g r a n u l e s v a r i e d f r o m 30 to 35%, w h i c h is
199
in a g r e e m e n t w i t h a n a l y s e s u s i n g o t h e r m e t h o d s a n d w h i c h s u p p o r t s the h y p o t h e s i s t h a t the v o l u t i n g r a n u l e s are a l m o s t ent i r e l y c o m p o s e d of p u r e p o l y p h o s p h a t e .
INTEGRATED IMAGE ANALYSIS AND ELECTRON E N E R G Y L O S S S P E C T R O S O C P Y IN E M W.C. de B r u i j n , C.W.J. S o r b e r , T. de J o n g and J.B. v a n D o r t * AEM Unit, Clin. Pathol. Inst. Erasmus University, PO Box 1738, Rotterdam; *Hogeschool West-Brabant, Lab Onderwijs, Etten Leur, The Netherlands
T h e c o m b i n a t i o n , Z e i s s E M 902 m i c r o s c o p e w i t h an I B A S - 2 0 0 0 i m a g e a n a l y s i s system, allows morphometrical quantitat i o n a n d i n t e g r a t i o n of i m a g e s f r o m e l e m e n t - r e l a t e d e l e c t r o n e n e r g y loss (EELS) w i t h c e l l - s t r u c t u r e - r e l a t e d inelastically scattered electron popul a t i o n s (ESI). C o n t r a s t t h r e s h o l d i n g has b e e n app l i e d to s u c h s p e c t r o s c o p i c a l i m a g e s to o b t a i n a p p r o p r i a t e a n d o b j e c t i v e segmentation. In g l u t a r a l d e h y d e - f i x e d cells, c h e m i c a l e l e m e n t s in two c e l l components have been morphologically analyzed: o n e i n t r i n s i c e l e m e n t a n d a c y t o chemical reaction product. It is s h o w n t h a t for the i n t r i n s i c n u c l e a r p h o s p h a t e and for a b a r i u m - c o n t a i n i n g r e a c t i o n p r o d u c t in the R E R ( f r o m an a r y l s u l f a t a s e r e a c t i o n ) a v o l u m e f r a c t i o n c a n be e s t i m a t e d . The use of an i o n - e x c h a n g e b e a d type of B i o - s t a n d a r d (Polaron, UK) has rec e n t l y b e e n a d v o c a t e d for e l e m e n t a l c o n c e n t r a t i o n e s t i m a t i o n s , in X - r a y m i c r o analysis. It has b e e n s h o w n t h a t a cons t a n t ratio, r e p r e s e n t i n g the r e l a t i v e concentration ( R ) of t h e s e s t a n d a r d s , c o u l d be o b t a i n e ~ . F o r e a c h t y p e of s t a n d a r d a p r o p o r t i o n a l i t y c o n s t a n t has b e e n c a l c u l a t e d , that relates R to the s t a n d a r d ' s m e a n (C) concentration value, estimated by neutron actlvatlon analysis. For quant i t a t i v e E E L S a n a l y s i s the c o n s t a n c y of R is i n v e s t i g a t e d for s t a n d a r d s c o n t a i n e ~ t h e r Ca, Co, Ni, Fe or Cu. Special a t t e n t i o n is g i v e n to d e l i b e r a t e l y i n t r o duced section thickness variations, to s h o w that, w i t h i n l i m i t s , the R v a l u e is i n d e p e n d e n t of s e c t i o n x thickness. X
.
.