QUANTITATIVE X-RAY MICROANALYSIS OF BIOLOGICAL CRYOSECTIONS DEPENDS ON ICE CRYSTAL DAMAGE

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QUANTITATIVE X-RAY MICROANALYSIS OF BIOLOGICAL CRYOSECTIONS DEPENDS ON ICE

CRYSTAL DAMAGE

K. Zierold

To cite this version:

K. Zierold. QUANTITATIVE X-RAY MICROANALYSIS OF BIOLOGICAL CRYOSECTIONS DE- PENDS ON ICE CRYSTAL DAMAGE. Journal de Physique Colloques, 1984, 45 (C2), pp.C2-447- C2-450. �10.1051/jphyscol:19842101�. �jpa-00223768�

30URNAL DE PHYSIQUE

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

QUANTITATIVE X-RAY MICROANALYSIS OF BIOLOGICAL CRYOSECTIONS DEPENDS ON ICE CRYSTAL DAMAGE

K. Zierold

Max-Planck-Institut fur Systemphysiologie, Rheinlanddamm 201, 4600 Dortmund, F.R.G.

Résumé - A l'aide de la microanalyse par rayons X sur des cryo- coupes d'épaisseur de 100 nm de standards gélatine-glycérol et de foie de rat on constate que le rapport pic sur fond (p/b) décroît avec l'augmentation de la taille des cristaux de glace.

Abstract - It is shown by X-ray microanalysis of 100 nm thick cryosections from glycerol-gelatine standards and rat liver that the measured peak-to-background ratio (p/b) decreases with increasing ice crystal size.

INTRODUCTION

X-ray microanalysis of freeze-dried cryosections is used to measure the distribution of elements in different compartments of biological cells and tissues (1-5). Ice crystal damage of the ultrastructure due to insufficient freezing velocity is a well known preparation artefact of this method (6). However, the displacement of elements, particular- ly of diffusible ions as sodium, chlorine, potassium and calcium, is assumed to take place in dimensions not larger than one ice crystal diameter. Therefore X-ray microanalysis by scanning an electron beam in an area larger than a few ice crystal diameters in size was expect- ed to be independent on freezing damage. The experiments described in the following contradict to this assumption.

MATERIALS AND METHODS

The cooling chain preparation method as described previously (7, 8) is used for the preparation of the cryosections. Two kinds of speci- men were studied:

1. Droplets of 20?o glycerol-gelatine and 80% electrolyte solution of known composition, e.g. KC1 varying in concentration between 3.1 and 200 mMol/liter. These specimens were used as standards for quantitative X,-ray microanalysis (9).

2. Freshly excised pieces of rat liver, about 1 mm in diameter.

100 nm thick cryosections, prepared by means of the Reichert FC4 cryoultramicrotome were transferred to the electron microscope under cold nitrogen gas atmosphere. Freeze-drying of the sections was enabled by evacuating the cryotransfer chamber and loosening the cold contact to the grid holder. X-ray microanalysis was performed in a Siemens Elmiskop ST 100 F, a scanning transmission electron micros- cope (STEM) with a field emission gun, operated at 100 kV, by means of an energy dispersive SiLi-detector (nuclear semiconductor) and a multichannel analyzer (Link Systems). The scanning area is varied from 278 nm x 444 nm to 6940 nm x 11100 nm, analysis time was 100 s.

Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:19842101

C2-448 J O U R N A L D E PHYSIQUE

T h e s e c t i o n s w e r e k e p t a t 1 3 8 K i n t h e e l e c t r o n m i c r o s c o p e . F o r t h e e v a l u a t i o n o f t h e X - r a y p e a k s p / b - v a l u e s w e r e c a l c u l a t e d b y d i v i d i n g t h e p e a k h e i g h t ( p ) t h r o u g h t h e m e a n X - r a y i n t e n s i t y b e t w e e n 4 . 5 a n d 5 . 5 keV ( b ) .

RESULTS

F i g . 1 s h o w s a c r y o s e c t i o n o f g l y c e r o l - g e l a t i n e m i x e d w i t h 2 0 0 m M o l / l KC1 w i t h t h e m e a n i c e c r y s t a l d i a m e t e r l e s s t h a n 5 0 nm. A c o r r e s p o n d - i n g X - r a y s p e c t r u m i s a d d e d ( F i g . 2 ) . F i g . 3 w a s o b t a i n e d a f t e r s l o w e r f r e e z i n g t h e s a m e s p e c i m e n t y p e a s i n F i g . 1 . T h e m e a n i c e c r y s t a l d i a m e t e r i s c a . l / u m . F i g . 4 s h o w s t h e c o r r e s p o n d i n g X - r a y s p e c t r u m . p / b - v a l u e s f o r s u l f u r a n d p o t a s s i u m , d e r i v e d f r o m X - r a y s p e c t r a o f c r y o s e c t i o n s o f g l y c e r o l - g e l a t i n e m i x e d w i t h KC1 a r e s k e t c h e d i n F i g . 5 a n d 6 , r e s p e c t i v e l y . T h e m a i n r e s u l t i s t h a t t h e p / b - v a l u e d e c r e a s e s w i t h i n c r e a s i n g i c e c r y s t a l d i a m e t e r . T h i s h o l d s f o r d i f f u s i b l e i o n s ( e . g . p o t a s s i u m ) a s w e l l a s f o r b o u n d e l e m e n t s ( e . g . s u l f u r ) . S i m i l a r r e s u l t s a r e o b t a i n e d i n b i o l o g i c a l s p e c i m e n s . F i g . 7 s h o w s r a t l i v e r w i t h i n t e r m e d i a t e i c e c r y s t a l s i z e . T h e p / b - v a l u e o f p h o s p h o r u s d e p e n d i n g o n t h e i c e c r y s t a l s i z e i n t h e c y t o p l a s m a n d t h e n u c l e u s i s d r a w n i n F i g . 8.

F i g . 1

-

F i g . 2 - X - r a y s p e c t r u m o f t h e c r y o s e c t i o n i n F i g . 1 . S c a n n i n g a r e a = 6 9 4 nm x 1 1 1 0 nm.

F i g . 3

-

F i g . 4 - X - r a y s p e c t r u m o f t h e c r y o s e c t i o n i n F i g . 3 . S c a n n i n g a r e a = 6.941um x I l . l / u m .

sulfur in glycerol-gelotine potossium in glycerol gelotime

mixed with mixed with

200 mMolll KC1 ^*-• 200 mMolll KC1

.-.

6.25 mMolll KC1 - - - - ^{- a} 100 mMolll KC1

.-

- -.

10 6.25 mMolll KC1

..

^..

ice . . .

.

.

diometer (nmf 1 diameter (nm)

F i g . 5 - p / b o f s u l f u r v e r s u s m e a n i c e c r y s t a l d i a m e t e r . S.E.M. o f m e a s u r e d p o i n t s = + 8 % .

F i g . 6 - p / b o f p o T a s s i u m v e r s u s m e a n i c e c r y s t a l d i a m e t e r . S.E.M. o f m e a s u r e d p o i n t s = 2 107;.

phosphorus in rot liver cytoplasm ^.-a

\

^.-

^-.

L meon ice crystal diometer fnm)

I

50 100 200 300 LOO

F i g . 7 - C r y o s e c t i o n o f r a t l i v e r o f i n t e r m e d i a t e i c e c r y s t a l s i z e . C = c y t o p l a s m ; N = n u c l e u s ; b a r = l / u m .

F i g . 8 - p / b o f p h o s p h o r u s i n t h e c y t o p l a s m a n d n u c l e u s o f r a t l i v e r , d e p e n d e n t o n t h e m e a n i c e c r y s t a l s i z e . S.E.M. o f m e a s u r e d p o i n t s = + 10:;.

-

DISCUSSION

T h e s e r e s u l t s c o u l d b e e x p l a i n e d a s f o l l o w s : I n b i o l o g i c a l m a t e r i a l o f l o w d e n s i t y a 1 0 0 k e V - e l e c t r o n h a s a m e a n f r e e p a t h o f a b o u t 1 0 0 nm.

T h e m e a n d i s t a n c e b e t w e e n c h a r a c t e r i s t i c a t o m s h o m o g e n e o u s l y d i s t r i - b u t e d w i t h a v o l u m e d e n s i t y o f 1 0 0 m M o l / l = 6 2 0 0 0 a t o m s i n a c u b e o f 1 0 0 nm l e n g t h o f s i d e i s a b o u t 2 . 5 nm. P r o j e c t e d t o t h e s e c t i o n s u r f a c e t h e i n t e r a t o m i c d i s t a n c e a s s e e n f r o m t h e i m p i n g i n g e l e c t r o n s i s 0 . 4 nm. A f t e r p r e c i p i t a t i o n o f i c e c r y s t a l s d u r i n g c r y o f i x a t i o n t h e d e n s i t y o f t h e r e m a i n i n g m a t e r i a l a c c u m u l a t e d b e t w e e n t h e i c e c r y s t a l s i s e n h a n c e d , a n d t h e i n t e r a t o m i c d i s t a n c e b e t w e e n t h e c h a r a c t e r i s t i c a t o m s i s r e d u c e d a s s c h e m a t i c a l l y d r a w n i n F i g . 9 . R e l a t i v e l y s m a l l i c e c r y s t a l s a s s k e t c h e d i n F i g . 9 b r e s u l t i n s m a l l i o n d i s p l a c e m e n t s ,

C2-450 JOURNAL DE PHYSIQUE

w h e r e a s l a r g e r i c e c r y s t a l s c o m p r e s s t h e i o n s i n m a t e r i a l w a l l s o f e n h a n c e d d e n s i t y ( F i g . 9 c ) . A s a c o n s e q u e n c e e l e c t r o n s h i t t i n g h o l e s l e f t a f t e r i c e s u b l i m a t i o n p e n e t r a t e t h e s e c t i o n w i t h o u t i n t e r a c t i o n w i t h t h e s p e c i m e n . E l e c t r o n s h i t t i n g t h e m a t e r i a l w a l l s a r e s c a t t e r e d p r e f e r a b l y b y t h e a t o m s i n t h e u p p e r p a r t o f t h e s e c t i o n , a n d t h e e x c i t a t i o n p r o b a b i l i t y o f t h e a t o m s b e l o w i s r e d u c e d . T h i s e f f e c t r e s u l t s i n r e d u c e d p / b - v a l u e s d e p e n d i n g o n t h e i c e c r y s t a l s i z e .

9

a ^{o o F i g . 9} - S c h e m a t i c d r a w i n g o f i o n d i s - o o p l a c e m e n t s i n c r y o s e c t i o n s d u e t o i c e

0 0

0 O c r y s t a l g r o w t h a s s e e n f r o m t h e s i d e . o C i r c l e s = i o n s , h a t c h e d a r e a s = i c e

c r y s t a l s . a ) T h e i o n d i s t r i b u t i o n i s a s s u m e d t o b e s t a t i s t i c a l l y h o m o g e n e o u s

b ^{i n a} s e c t i o n w i t h o u t i c e c r y s t a l s .

b ) S m a l l i c e c r y s t a l s c a u s e s m a l l i o n d i s p l a c e m e n t s . T h e i o n s a r e s t i l l homo- g e n e o u s l y d i s t r i b u t e d . c ) L a r g e i c e c r y s t a l s a c c u m u l a t e t h e i o n s i n n a r r o w m a t e r i a l w a l l s . T h e i o n d i s t r i b u t i o n b e c o m e s i n h o m o g e n e o u s .

C

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373

8 5 .

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1 9 8 1 / I I ( 1 9 8 1 ) 4 5 5 .

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