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T I S S U E & C E L L 1 9 81 1 3

4 ) 6 2 3 - 6 4 3 0 0 4 0 - 8 1 6 6 / 8 1 /0 0 4 8 0 6 2 3 5 0 2 . 0 0

1 9 81 L o n g m a n G r o u p L t d .

B R Id L G U P T A a nd T H E O D O R E A H A L L

T H E X R A Y M I C R O A N A L Y S I S O F

F R O Z E N H Y D R A T E D S E C T I O N S IN S C A N N I N G

E L E C T R O N M I C R O S C O P Y : A N E V A L U A T I O N

A B S T R A C T . T h e p r e se n t s ta t u s o f t h e te c h n i q u e t o m e a s u r e c o n c e n t r a t i o n s o f e l e c tr o -

l y t e e l e m e n t s a n d d r y m a s s i n ,-. 1 t~ m t h ic k f r o z e n - h y d r a t e d s e c t i o n s o f s o f t b i o l o g i c a l

t is s u e s w i th e l e c t r o n p r o b e X - r a y m i c r o a n a l y s i s i n a s c a n n i n g e l e c t r o n m i c r o s c o p e is

c r i t ic a l l y r e v i e w e d . T h e t e c h n i q u e i s t o q u e n c h - f r e e z e f r e sh s p e c i m e n s t o < - 1 8 0 C ,

e t a , -. I tL m t h i c k h y d r a t e d c r y o s e c t i o n s ( ~ < - - 7 0 C ) , t r a n s f e r o n t o a c o l d s t a g e

( < - - 1 7 0 C ) o f a s u i ta b l e m i c r o a n a l y t ic a l a r r a n g e m e n t , o b t a i n s c a n n i n g t ra n s -

m i s s i o n i m a g e s t o i d e n t i f y t h e c e ll a n d t i s s u e c o m p a r t m e n t s , l o c a t e a n e l e c t r o n p r o b e

( s e v e r a l p .m -" o 1 00 n m ) o n t h e a r e a s o f i n t e r e s t a n d c o l l e c t X - r a y q u a n t a . T h e X - r a y

q u a n t a a r e c o l le c t e d w i t h s u i ta b l e s p e c t r o m e t e r s ( W D S a n d E D S ) a n d p r o c e s se d w i t h a

c o m p u t e r u s in g a c o m p r e h e n s i v e p r o g r a m m e b a s e d o n c o n t i n u u m n o r m a l i z a t i o n p r o -

c e d u r e s ( ' H a l l ' p r o g r a m m e ) . T h e c r y o s e c t i o n s a r e a n a l y s e d f i r st i n a h y d r a t e d s t a t e a n d

s e c o n d a f t e r d e h y d r a t i o n w i t h in t h e m i c r o a n a l y s er c o l u m n t o o b t a i n d i r e c tl y e l e m e n t a l

c o n c e n t r a t i o n s i n m M k g - 1 w e t w t a n d m M k g -1 d r y w t o f t h e c o m p a r t m e n t s

i d e n t i f i e d u n d e r t h e b e a m . T h e l o c a l w a t e r - f r a c t i o n s a r e e s t i m a t e d a n d t h e e l e m e n t a l

c o n c e n t r a t i o n s c o n v e r t e d i n t o m M 1 t w a t e r . I n t h e p a s t 7 y e a r s th e t e c h n i q u e h a s

b e e n a p p l i e d t o o b t a i n f u l ly q u a n t i t a t i v e i n f o r m a t i o n o n N a , K , C I , P , S . C a a n d H _ -O

i n m o r e t h a n t e n t y p e s o r t i s s u e .

I n l r o d u c t i o n

T H E e l e c t r o l y t e s o f c h e m i c a l e l e m e n t s ,

e s p e c i a l l y N a + , K + , C I - , C a ~+ a n d M g 2 + a r e

w i d e l y i m p l i c a t e d i n a v a r i e t y o f c e l l - fu n c t i o n s

( C i v a n , 1 9 78 ) . F o r a p r o p e r a s s e s s m e n t o f

t h e i r r o l e o n e n e e d s t o k n o w t h e q u a n t i t a t i v e

d i s t r i b u t i o n o f t h e s e e l e c t r o l y t e s i n d i f f e r e n t

s u b c o m p a r t m e n t s i n a n d a r o u n d t h e c e i ls

a t t h e m i c r o s c o p i c l e v el . T h e e l e c t r o n p r o b e

X - r a y m i c r o a n a l y s i s ( E P X M A ) o f t is su e

s e c t i o n s p o t e n t i a l l y p e r m i t s su c h m e a s u r e -

m e r i ts t o b e m a d e i n a r e l i a b le , f u l l y q u a n t i t a -

: a n d n o n - d e s t r u c t i v e w a y ( H a l l , 1 9 71 ) .

. ; : .p e n d i n g o n t h e t is s u e p r e p a r a t i v e a n d

i n s t r u m e n t a l a p p r o a c h , a n a n a l y t i c a l s p a t i a l

r e s o l u t i o n o f b e t t e r t h a n 1 0 0 r i m w i t h a

s e n s i t iv i t y o f a f e w m M i s a l r e a d y a r o u t i n e

D e p a r t m e n t o f Z o o l o g y , B i o l o g i c a l M i c r o p r o b e

L a b o r a t o r y , C a m b r i d g e U n i v e r s i t y , D o w n i n g S t r e e t,

C a m b r i d g e C B 2 3 E J , U K .

A d d r e s s f o r c o r r e s p o n d e n c e : D r B r i j L . G u p t a ,

Z o o l o g y D e p a r t m e n t , D o w n i n g S t r e e t , C a m b r i d g e

C B 2 3 E J , U K .

Rec e i ved 25 Ap r i l 1981.

Rev i sed 15 Ju l y 1981 .

4 0

a c h i e v e m e n t ( f o r a r e c e n t r e v i e w s ee H a l l ,

9 7 9 a ) .

F o r d i f fu s i b l e e l e m e n t s t h e m o s t r e l i a b l e

a p p r o a c h is t o q u e n c h - f r e e z e c el ls a n d t is s u e s

i n a k n o w n p h y s i o l o g i c a l s ta t e a n d c u t

c r y o s e c t i o n s e i t h e r u l t r a t h i n ( < 2 0 0 n m t h i c k )

o r t h i ck ( > 5 0 0 r i m ) . T h e c r y o s e c t i o n s c a n

e i t h e r b e c a r e f u l l y f r o z e n - d r i e d p r i o r t o

E P X M A ( A p p l e t o n , 1 9 78 ; B a r n a r d a n d

S e v 6 u s , 1 9 78 ; C a m e r o n

e t a L

1 9 7 9 ; D / S r g e

e t a L

1 9 7 8 ; S o m l y o

e t a L

1 9 7 7 ) o r th e y

m a y b e a n a l y s e d f i r s t i n a f r o z e n - h y d r a t e d

s t a t e a n d s u b s e q u e n t l y a f t e r a c o m p l e t e

d e h y d r a t i o n w i t h i n t h e c o l u m n o f t h e e l e c t r o n

m i c r o s c o p e ( E M ) u n d e r h i g h v a c u u m ( B a c -

a n e r e t a L 1 9 73 ; H u t c h i n s o n e t a L 1 9 7 4 ,

1 9 7 8; M o r e t o n e t a L 1 9 7 4 ; G u p t a , 1 9 7 6 ,

1 9 7 9 ; G u p t a

e t a L

1 9 7 7 , 1 9 7 8 a , b , c ; G u p t a

a n d H a l l , 1 9 7 9 , 1 9 8 1 a , b , 1 9 8 2 ; C i v a n

e t a L

1 9 8 0 ; D o w

e t a l .

1 98 1 ; L u b b o c k

e t a L

1 9 8 I ;

R o s s e t a L 1 9 81 ; S a u b e r m a n n e t a L 1 9 8 1 a ,

b ; Z i e r o l d , 1 9 8 2 ). T h e a n a l ys i s o f c r y o -

s e c t i o n s i n t h e f r o z e n - h y d r a t e d s t a te p e r m i t s

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

d i r e c t l y i n m M p e r k g w e t m a s s o f t il e s p e c i -

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u n s o u n d ( s e e b e l o w f o r d e ta i ls ) a n d l h e s p ec i -

m e n s a n a l y s e d i n th e s e s t u d i e s m u s t h a v e

b e e n s u b s t a n t i a l l y d e h y d r a t e d ( G u p t a

et aL,

1 97 7, 1 9 8 0 a) . A l t h o u g h o t h e r w o r k e r s h a v e

d e v e l o p e d t e c h n i q u e s f o r t h e a n a l y s i s o f

f r o z e n - h y d r a t e d s e c t i o n s ( B a c a n e r

et aL,

1 9 7 3 ; H u t c h i n s o n

et aL,

1 9 7 4 ; R o s s

et at.,

1 9 81 ) a n d b u l k t i ss u e s ( L e c h e n e

et aL,

975 ;

F u c h s

et aL,

1 9 7 8 ; M a r s h a l l , 1 9 8 0) in t h e

S E M , w e a p p e a r t o b e t h e o n l y g r o u p t o

h a v e p u b l i s h e d e x t e n s i v e b i o l o g i c a l r e s u l t s

f r o m t h e d i r e ct a n a l ys i s o f a p p r o x i m a t e l y

t~ tn l t h i c k f r o z e n - h y d r a t e d s e c t i o n s .

R e c e n t l y S a u b e r m a n n et aL ( 1 9 8 1 a , b )

h a v e p u b l i s h e d s o m e ' n e w ' a n d ' p r ac t i ca l '

t e c h n i q u e s f o r t h e m i c r o p r o b e a n a l y s i s o f

0 . 5 -1 /~ nl t h i c k f r o z e n - h y d r a t e d s e c t i o n s

i n a n S E M f i tt e d w i t h E D S , t o g e t h e r w i t h

s o m e i l lu s t r a t i v e d a t a f r o m r a t r e n a l p a p i l l a

t i s s u e ( B u l g e r

et aL,

1 98 1) , H o w e v e r t h e i r

t e c h n i q u e s a r e n o t n e w a n d t h e i r a rt i c le s d r a w

l i b er a ll y o n o u r p u b l i s h e d o b s e r v a t i o n s , o f t e n

w i t h o u t c i t a t i o n . T h e d e s c r i p t i o n o f t l i e i r

t e c h n i q u e s a n d r e s u l t s c o n t a i n s s e r i o u s

a n o m a l i e s . T h e i r i l lu s t r a ti v e b i o l o g i c a l r e s u lt s

( B u l g e r

et aL,

1 98 1 ; S a u b e r m a n n a n d R i l e y ,

1 9 8 0 ) w e r e o b t a i n e d b y e l e m e n t a l a n a l y s i s

o f

fi ozen-dried

a n d n o t f r o z e n - h y d r a t e d

s e c t i o n s . N e v e r t h e l e s s , i n t h e i r g e n e r a l i z e d

s u m m a t i o n ( S a u b e r m a n n

el aL,

1 9 8 1 a , p .

2 6 1 ) t h e y d i s m i s s o u r t e c h n i q u e s b y s t a t i n g :

A l t h o u g h t h e s e t e ch n i q u e s c a n a n a l y se

f r o z e n - h y d r a t e d s e c t i o n s t w o m a j o r p r o b l e m

a r e a s h a v e p r e c l u d e d r e li a b le a n d g e n e r a l

a p p l i c a t i o n . F i r s t , t h e c r y o s e c t i o n i n g p r o c e s s

w a s p o o r l y u n d e r s t o o d a n d i t w a s i m p o s s i b l e

t o c u t u n i f o r m , f u l l y h y d r a t e d s e c t i o n s

c o n s i s t e n t l y w i t h a v a i la b l e s e c t i o n i n g a p p a -

r a tu s . S e c o n d , t h e r e w a s n o s i m p l e s y s t em

f o r s p e c i m e n s u p p o r t , t r a n s fe r a n d m a i n -

t e n a n c e a t l o w t e m p e r a t u r e w i t h i n t h e i n s t ru -

m e n t (1 0 ). T h e s e p r o b l e m s a p p e a r t o h a v e

b e e n s o l v e d b y t h e t e c h n i q u e s d e s c r i b e d h e r e ;

( r e f e r e n c e ( 1 0 ) is t o H a l l , 1 9 7 9 a ) .

I n t h e f o l l o w i n g a c c o u n t w e e x a m i n e t h e s e

s t a t e m e n t s a n d c r i t i c a l l y e v a l u a t e t h e " n e w "

t e ch n i q u es o f S a u b e r m a n n

et al.

G e n e r a l O u t l in e o f O u r M e t h o d s

ht ritro

p h y s i o l o g i c a l p r e p a r a t i o n ( G u p t a ,

1 9 7 6 ; G u p t a

et aL,

1976, 1978a , b , 1980b;

C i v a n

et aL,

1 9 8 0 ; G u p t a a n d H a l l , 1 9 8 1 a ) ,

o r b y f r es h e x c i si o n f r o m a n a n i m a l ( G u p t a

et aL,

1 9 8 0 b ; L u b b o c k

et aL,

1 9 8 ; D o w

et

aL,

1 9 8 1) , o r m a y c o n s i s t o f c e ll s il l s u s -

p e n s i o n ( J o n e s

et aL,

1 9 7 9 ) o r i n t i s s u e

c u l t u r e . S p e c i a l c a r e i s t a k e n t o ' a v o i d m o -

m e n t a r y c h a n g e s i n t h e l i v i n g t i s s u e d u e t o

e v a p o r a t i v e l o s s o f w a t e r b e f o r e q u e n c h -

f re e z in g ( G u p t a

et aL,

1 9 77 ; H a l l a n d G u p t a ,

1 9 8 2) . T h e f i 'o z e n t i s su e is s t o r e d u n d e r

l i q ui d n i t ro g e n ( - 1 9 6 C ) , t o b e s e c t io n e d ,

w h e n c o n v e n i e n t , a t a b o u t 1 t ~m a t t e m p e r a -

t u r es u s u a ll y b e l o w - 7 0 C e i t h e r in a

c r y o s t a t ( S L E E , L o n d o n ; A p p l e t o n , 1 9 78 )

o r i n a m o d i f i e d C r y o K i t ( L K B , S w e d e n ) .

T h e s e ct io n s ar e i m m e d i a te l y m o u n t e d o n

a l u m i n i u m - c o a t e d n y l o n f il m t i g h tl y s tr e t c h e d

o v e r a 2 - 3 m m h o l e in a D u r a l i u m h o l d e r

a n d t r a n s f e r r e d t o a l i q u i d n i t r o g e n ( L N e )

c o o l e d 'p a r k i n g ' a n d ' s t o r a g e ' D e w a r w h e r e

t h e y c a n b e s a f e l y l ef t, i f n e c e s s a r y f o r s e v -

e ra l w e e k s , ill a d r y a t m o s p h e r e < - 1 9 0 C

w i t h o u t a n y a p p r e ci a b l e g a i n o r l o ss o f

w a t e r . W h e n c o n v e n i e n t , t h e s e c t io n h o l d e r s

a r e l o a d e d t h r o u g h a n e v a c u a t e d a i r - l o c k

o n t o t h e c o l d ( - 1 7 0 C ) t r a n s m i s s i o n s t a g e

( T a y l o r a n d B u r g e s s , 1 9 77 ) o f a J E O L

J X A - 5 0 A S E M - m i c r o a n a l y s e r . I n t h e m i c r o -

a n a l y s er c o l u m n t h e s p e c i m e n a n d t h e st a g e

a r e p r o t e c t e d f ro m c o n t a m i n a t i o n b y a l ar g e

c o l d - sh i e l d ( - 1 9 0 C ) p o s i t i o n e d a b o u t 1 m m

a b o v e t h e s p e c i m e n . T h e m i c r o a n a l y s e r

o p e r a t e s w i t h a c c e l e r a t i n g v o l t a g e u p t o

5 0 k V a n d h a s b e e n f i tt ed w i t h a K E V E X -

L I N K E D S a n d L I N K m u l t i c h a n n e l a n a ly s e r

b a c k e d w i t h a n o n - li n e c o m p u t e r u s i n g a

c o m p r e h e n s i v e " H a ll ' p r o g r a m f o r X -r a y

d a t a r e d u c t i o n ( L i n k S y s t e m s , H i g h

W y c o m b e , B u c k s ., U K ) . A d d i t i o n a l l y t w o

f u ll y f o c u s i n g W D S a r e f it t ed t o a n a l y s e v e r y

l o w c o n c e n t r a t i o n s o f i n tr a c e ll u l a r N a a n d

C a (i n t h e p r e s e n c e o f h i g h c o n t i n u u m

a n d h i g h K ) w h e r e E D S m a y b e u n a b l e t o

c o p e . I n I tL m t h i c k f u ll y h y d r a t e d s e c t i o n s

a t e s te d a n a l y t i c a l s p a t i a l r e s o l u t i o n o f 0 .2

t~ nl a n d a d e t e c t i o n l im i t o f a b o u t 1 r n M a r e

r e g u l a r l y a c h i e v e d .

O u r p r o c e d u r e s t a rt s w i t h t h e q u e n c h - f r e e z i n g C o m p a r i s o n o f echniques

o f l i v in g t i ss u e i n m o n o c h l o r o t r i f l u o r o -

m e t h a n e ( F r e o n - 1 3 o r l s c e o n - I 3 , H R P I . C r y o p r e s e rv a t i o n o f t i ss u e

S a le s L t d , L o n d o n ) o r i n s o m e o t h e r c r y o g en . G o o d c r y o p r e s e r v a t i o n o f l i v i n g c ei ls a n d

T h e l i v i n g t i s s u e m a y b e o b t a i n e d f r o m a n t is s ue s w i t h a m i n i m u m o f i c e- c ry s ta l d a m a g e

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is g e r m a n e tO a ll c r y o t e c h n i q u e s i n m i c r o -

s c o p y i n c l u d i n g t h e f r e e z e - f i - a c t u r e m e t h o d s .

H o w e v e r f o r t h e m i c r o p r o b e a n a l y s i s o f

t h i c k s e c t i o n s i n S E M w h e r e t h e i m a g e

r e s o l u t i o n i s n o t b e t t e r t h a n a b o u t 0 "2 t m L

t he ~ p res ence o f i ce c ry s t a l s can be t o l e ra t ed

p r o v i d e d i t d o e s n o t c a u s e r e d i s t r i b t ~ t i o n o f

d i f f u s ib l e e l e m e n t s o v e r d i s t a n c e s l a r g e r th in 1

t h e s p a t i a l r e s o l u t i o n o f th e a n a l y s e s . F o r

m i c r o a n a l y t i c a l w o r k t h e u s e o f c h e m i c a l

f i x a t i v e s a n d p e n e t r a t i n g c r y o p r e s e r v a t i v e s

( e . g . g l y c e r o l ) i s n o t p e r m i s s i b l e ( H a l l ,

1 9 7 9a ) . F u r t h e r m o r e , l a r g e p i e c e s o f t is s u e

a r e o f t e n r e q u i r e d f o r d e t a i l e d a n a l y s e s o f

s e v e r al t is s u e c o m p a r t m e n t s i n t h e s a m e

s e ct io n s. T h e r e f o r e t e c h n i q u e s s u c h a s s la m -

f r e e z i n g ( H e u s e r

e t a L

1 9 7 9 ) w h i c h m a y

p r o v i d e e x c e l le n t m o r p h o l o g i c a l p r e s e r v a t i o n

i n a 1 0 - 2 0 t m a t h i c k s u r f a c e z o n e a r e l es s

s u i t a b l e t h a n i m m e r s i o n - f r e e z i n g i n l i q u id

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

a n d i o n i c r e d i s t r i b u t i o n h a s b e e n d i s c u ss e d

i n s o m e d e t a il e l s e w h e r e ( G u p t a e t a l .

1 9 7 7 ; A p p e n d i x i n G u p t a e t a k 1980b).

( i) I n o u r l a b o r a t o r y s a t i s f a c t o r y p r e s e r -

v a t i o n o f e v e n l a r g e s p e c i m e n s s u c h a s h t

v i t r o p r e p a r a t i o n s o f t o a d u r i n a r y b l a d d e r

( C i v a n e t a l . 1 98 0) a n d r a b b i t i l e u m ( G u p t a

e t a L I 9 7 8 b ) i s a c h i e v e d b y u s i n g a w e l l -

s t i r r e d , s u p e r - c o o l e d ( S o m l y o e t a l . 1977)

v o l u m e o f F r e o n -1 3 ( t e m p e r a t u r e a b o u t

- 1 9 0 C ) . T o a c h i e v e a m o r e re p r o d u c i b l e

q u a l i t y o f c r y o p r e s e r v a t i o n " w e h a v e n o w

b u i l t a q u e n c h i n g d e v i c e i n w h i c h t h e c r y o -

g e n v e ss e l is tr i g g e r e d t o r i se a t a p r e d e t e r -

m i n e d s p e e d u p t o t h e t is s u e ( B a r n a r d , 1 98 2 ;

G u p t a a n d H a l l , 1 98 2). F i g s . 1 -1 4 p r o v i d e

e x a m p l e s f r o m a n u m b e r o f t is su e s to s h o w

t h a t t h e c r y o p r e s e r v a t i o n i s f r e e f r o m i c e -

c r y s t a l s v i s i b l e a t t h i s r e s o l u t i o n , a l t h o u g h

p a r a l l e l s t u d i e s w i t h f r e e z e - s u b s t i t u t i o n u s i n g

t h e m e t h o d o f H e u s e r a n d R e e s e ( 19 7 6) d o

r e v e a l i c e c r y s t a l s , m e a s u r i n g 0 .0 5 -- 0 .2 tz nl

i n d i a m e t e r i n t h e s e s p e c i m e n s . O u r s t u d i e s

o n a v a r i e t y o f b i o l o g ic a l t is s ue s i n c l u d i n g

d e l i c a t e i n se c t e p i t l e l i a w i t h f r e e ,f l u id

s p a c e s h a v e g iv e n n o e v i d e n c e o f i o n ic

r e d i s t r i b u t io n e i t h e r i n s id e o r o u t s i d e t h e

c e ll s. I o n i c g r a d i e n t s i n n a r r o w e x t r a c e l l u l a r

s p a c e s w i t h l it tl e o r g a n i c m a t r i x h a v e b e e n

m e a s u r ed ( G u p t a

e t a L

1 9 7 8 a , b ; 1 9 8 0 b ;

G u p t a a n d H a l l , 1 9 7 9 , 1 98 a ) .

( i i ) S a u b e r m a n n e t a l . ( 1 9 8 1 a , b ) q u e n c h -

f re e z e t h e i r sp e c i m e n s e i t h e r m o u n t e d o n

l a rg e C o p p e r , c h u c k s ( 4 12 m m ) o r u n -

G U P T A A N D H A L L

m o u n t e d ( B u l g e r e t a l . 1981) i n m e l t i ng

F r e o n - 1 2 ( - - 15 8 C ). T h e i r s p e c i m e n s a p p e a r

t o b e p o o r l y p r e s e r v e d w i t h i c e c r y s t a l s a s

l a rge a s 0 , 4 tLm eve n i n s a l t s o l u t i ons con -

t a i n i n g 2 0 ~ p o l y v in y l p y r r o l i d o n e ( a n

" e x c el le n t ' c r y o p r o t e c t a n t : S k a e r

e t a i .

1977)

wh i l e t he i ce c ry s t a l s if i t he i r b i o l og i ca l

s a m p l e s a l: e m u c h l a r g e r ( F i g s . 1 0 D a n d I 1 B

i n S a u b e r m a n n e t a l. , 1 9 8 1a , a n d F i g . 2 B

m B u l g e r e t a L 1981) . S uch ex t ens i ve i ce -

c r y s t a l d a m a g e ( a ) m a k e s g o o d c r y o s e c t i o n -

i n g d if f ic u l t a t l o w t e m p e r a t u r e , ( b ) p r o m o t e s

d e h y d r a t i o n o f s e c ti o n s , a n d ( c ) w il l d i s r u p t

s u b c e l h d a r or g a n el le s a n d m e m b r a n e b a r r i e r s

a n d t h e r e f o r e c a u s e r e d i s t r ib u t i o n o f h i g h l y

m o b i l e i o n s e s p e c ia l ly o f N a , K + a n d C I - .

T h e t e c h n i q u e s o f c r y o p r e s e r v a t i o n u s e d b y

S a u b e r m a n n

e t a L

a r e , t o s a y t h e l e a s t ,

i n d i f f e r e n t a n d d o n o t i n s p i r e c o n f i d e n c e

e v e n i n t h e i r t o w - r e s o l u t i o n ( 1 - 2 t ~ m )

a n a l y s e s ( a l s o s e e s e c t i o n 1 I b e l o w ) .

11 . C ryos ec f i on i ng

S a u b e r m a n n e t a k ( 1 9 8 1 a , p . 2 6 t ) c l a i m t h a t

it w a s i m p o s s i b le t o c u t s m o o t h c r y o s e c t i o n s

w i t h t h e a v a i la b l e s e c t io n i n g a p p a r a t u s a n d

t h e i r n e w t e c h n i q u e s h a v e o p e n e d u p t h e w a y

t o o b t a i n u n i f o r m l y s m o o t h f u ll y h y d r a t e d

s ec t i ons .

( i) O u r c r y o s t a t f o r c u t t i n g I t~ nl t h i c k c r y o -

s e c t io n s a t t e m p e r a t u r e s o f a b o u t - 8 0 C w a s

d e s i g n e d o n t h e p r e m i s e s ( e. g . S t u m p f a n d

R o t h , 1 96 5) t h a t ( a) t h e l a r g e c r y o c h a m b e r

( s iz e a b o u t 0 . 6 x 0 -6 x 0 . 4 m ) , s p e c i m e n a n d

k n i f e s h o u l d h a v e t h e s a m e s t a b l e t e m p e r a -

t u r e , ( b ) c a r b o n s t e e l k n i v e s w i t h l o w ( 1 2 - 1 4 )

b e v e t a r e b e t t e r t h a n g l a s s k n i v e s , ( c ) t h e c u t -

t i n g s p e e d s h o u l d b e a s l o w a s p o s s i b l e w i t h a

m o t o r - d r i v e n m i c r o t o m e f o r s m o o t h c y c li n g ,

a n d ( d ) a n a n t i r o l l p l a t e s h o u l d b e u s e d . T h i s

c r y o s ta t , c u s to m - b u i l t b y S L E E ( L o n d o n ) , h a s

f u n c t i o n e d s i n c e 1 9 72 , h a v i n g b e e n u s e d b y ,

a m o n g o t h e r s , S a u b e r m a n n a n d E c h l in ( 19 7 5)

a s p a r t o f t h e i r ' f i r m a n d w e l l- t e st e d t e c h n i q u e ' .

A l l t h e d e s i g n f e a t u r e s h a v e a l s o b e e n u s e d

f o r m a n y y e a r s b y o t h e r w o r k e r s , f o r e x a m p l e

D 6 r g e e t a l . ( I975, 1978) t o c u t I -_ 9 ~ m t h i c k

c r y o s e c ti o n s f o r E P X M A . T h e s p e c i m e n f o r

c r y o s e c t i o n i n g is g e n e r a l l y f r o z e n i n d r o p l e t s

o f t h e b a t h i n g m e d i u m ( R i n g e r , s e a w a t e r ,

b o d y f lu i d , e t c ) o n t h e t o p o f a t r u n c a t e d

c o p . p e r p i n ( d i a m e t e r a b o u t 1 .5 m m ) w h i c h

i s d i r e c t ly c l a m p e d i n t o t h e m i c r o t o m e c h u c k .

P i e c e s o f l a r g e r ti ss u e a r e m o u n t e d u n d e r

L N.% w i t h in a l o o p o f m a l l e a b le i n d i u m

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M I C R O A N A L Y S I S O F F R O Z E N - H Y D P , A T F D S P E C I M E N S 627

m e t a l c l a m p e d b y t w o f la t s u r f a c e s o f a

j a w - t y p e c h u c k ( C i v a n

et aL

1 9 8 0 ; R i c k

et aL 1 9 79 , 1 9 82 ). T h e l a t t e r a r r a n g e m e n t

p r o v i d e s a f i rm c l a m p i n g w i t h o u t c a u s i n g

i n t e r n a l f i ' a c t u r e i n t h e t i s s u e b l o c k .

M o r e r e c e n t ly o u r c ry o s t a t h a s b e e n m o d i -

f ie d to i n c o r p o r a t e L N.z c o o l i n g , a P o r t e r -

B l u m M a r k i m i c r o t o m e ( A p p l e t o n , 1 9 78 )

a n d t h e u s e o f l c m w i d e s e g m e n t s o f D -

p r o f i l e s t e e l k n i v e s ( R i c k et al. 1979) .

S m o o t h , f i ' o z e n - h y d r a t e d c r y o s e c t i o n s a r e

r e g u la r ly cu t w i t h o u r e q u i p m e n t a t t e m p e r a -

t u r e s d o w n t o - 9 5 C w i t h k n if e k e p t a f e w

d e g r e e s w a r m e r w i t h a k n i f e h e a t e r ( F i g s .

1 - 8) . T h e s e s e c t i o n s p r e s e r v e e x c e l le n t

m o r p h o l o g i c a l d e t a i ls i n a r a n g e o f ti s su e s

( F i g s. 9 - = 4 ; s e e o t h e r e x a m p l e s i n G u p t a

et aL

1 9 76 , 1 9 78 a , 1 98 0 b ; G u p t a a n d H a l l ,

1 9 8 1 b , 1 9 8 2 ; C i v a n

et al.

1 9 8 0 ; L u b b o c k

et aL 1 9 8 1 ; D e w et aL 1981) .

( i i ) S a u b e r m a n n et al. ( 1 9 7 7 a ) c a r r i e d o u t

a s t u d y o f t h e w o r k i n p u t a n d t h e q u a li t y

o f c r y o s e c t io n s c u t a t v a r i o u s t e m p e r a t u r e s .

T h e y c o n c l u d e d t h a t fo r cu t t i n g s m o o t h

c r y o s ec t io n s t h e e q u i p m e n t s h o u l d i n c o r p o -

r a te t h e p r i n c i p le s a - d m e n t i o n e d a b o v e .

S a u b e r m a n n et aL ( 1 9 8 1 a ) h a v e i n t r o d u c e d

a n e w c ry o a t t ac h m e n t f o r th e S O R V A L L

P o r t e r - B l u n t M a r k . I1 u l t r a t o m e w i t h a

h o l d e r f o r s i n g l e - e d g e d s a f e t y r a z o r b l a d e s .

H o w e v e r , th e y m a k e t h e a s s er t i o n t h a t e v e n

w i t h t h e i r n e w l y de si gn e .d e q u i p m e n t s m o o t h

c r y o s e c t i o n s

cannot

b e C ut be l o w - 4 0 C

a n d o n e s h o u l d p r e f er a b l y u s e a t e m p e r a t u r e

n e a r - 3 0 C . I t is t h e r e f o re n o t c l ea r w h y

t h e c r y o a t t a c h m e n t d e s i g n ed b y S a u b e r m a n n

et al. s h o u l d w o r k b e t te r th a n t h e e q u i p m e n t

a v a i l a b l e a n d i n u s e i n o t h e r l a b o r a t o r i e s .

( ii i) S u f f ic i e n tl y s m o o t h c r y o s e c t i o n s a r e

a l s o r e g u la r l y c u t at - 7 0 C o r b e l o w in

o t h e r l a b o r a t o r i e s ( A p p l e t o n , 1 9 74 ; F u c h s

et aL 1 97 8; M o n s o n , 1 9 8 t ; R i c k et aL

1 9 7 9 ; S o m l y o et aL 1 9 7 7 ; S i t t 6 e t a L , 1 9 8 0 ;

S e v 6 us , 1 9 7 8 ; T o r m e y a n d P l at z , I 9 7 9 ;

Z i e r o l d , 1 9 8 2 ). W i t h t h e e x c e p t i o n o f R i c k

et aL

( 1 9 7 9 ) a n d F u c h s

et al.

( 1 9 7 8 ) , a l l t h e s e

w o r k e r s c u t t h i n ( 1 0 0 -2 0 0 n m ) c r y o s e c t i o n s

w i t h g l a s s k n i v e s .

( i v ) S a u b e r m a n n e t a L ( 1 9 7 7 a , b , I 9 8 1 a )

a r g u e t h a t w h e n c r y o s e c ti o n s a r e c u t a t

t e m p e r a t u r e s b e lo w - 4 0 C t h e w o r k - l o ad

o n t h e k n i f e e d g e i s e n o r m o u s l y i n c r e a s e f f .

T h i s l o a d i s i n v er s e ly r e l at e d t o t h e c u t t i n g

t e m p e r a t u r e b u t is i n d e p e n d e n t o f t h e s e c ti o n

t h i c k n e ss . :T h e i n c r e a s e d w o r k , l o a d a t l o w

t e m p e r a t u r e s p r o m o t e s ' m u l t i p l e c h i p p i n g '

w h i l e ' s m o o t h c i l i p p i n g ' o n l y o c c u r s a t

- 3 0 C . T h e y f u r t h e r a r g u e t h at i f s m o o t h

c r y o s e c t io n s a r e o b t a i n e d a t - 7 0 C oi"

l o w e r , t i m e w o r k - l o f i d a c t u a l l y r a i s e s tim e

t e m p e r a t u r e a t t h e c u t t i n g fa ce t o - 3 0 C ;

t h e s e c t i o n s m a y a c t u a l l y m e l t i f c u t a t

- 8 0 C ( S a u b e r m a n n et aL 1981 b , p . 272) .

T h e a r g u m e n t s o f S a u b e r m a n n et aL

a r e b a s e d o n t h e a s s u m p t i o n t h a t al l t h e

p h y s i ca l w o r k n e e d e d f o r c r y o s e c t i o n i n g

p r o c e s s is c o n v e r t e d i n t o h e a t a t t h e c u t t i n g

i n te r fa c e . W h i l e s o m e r is e in t e m p e r a t u r e

d u e t o f r i c t io n a l h e a t m i g h t o c c u r , t h e r e i s

s t r o n g e x p e r i m e n t a l e v i d e n c e t h a t i t d o e s n o t

r ai se t h e t e m p e r a t u r e o f t h e c r y o s e c t io n

e v e n b y a f e w d e g r e e s ( S o m l y o , 1 9 81 ;

F r e d e r i c k a n d B u s i n g , 1 9 8 1 ) . I n a n e l e g a n t

e x p e r i m e n t S o m l y o (1 9 8 1) h a s c u t 1 0 0 - 2 0 0

n m t h i c k s ec t i o n s o f f ro z e n t o l u e n e ( m e l t i n g

p o i n t - 9 5 C ) i n t h e i r L K B C r y o K i t ( S o m l y o

et aL I 9 77 ) . S h e f o u n d t h a t s e c t io n s c o u l d

n o t b e o b t a i n e d w i t h t h e t e m p e r a t u r e o f

t h e g l a s s k n i f e a t - 9 4 C b u t t h a t s m o o t h

a n d s t a b l e fi a t s e c t i o n s w e r e o b t a i n e d w i t h

a k n if e t e m p e r a t u r e o f - 9 6 C .

T h e r e l e v a n t q u e s t i o n t h e r e f o r e i s w h y

o t h e r w o r k e r s c a n c u t s m o o t h c r y o se c ti o n s ,

b o t h t h i n a n d t h i c k , a t t e m p e r a t u r e s o f

< - 4 0 C w h i le S a u b e r m a n n a n d c o ll e ag u e s

a p p a r e n t ly c a n n o t ? I t c a n n o t b e d u e t o t h e

a l l e g e d h i g h e r ' h e a t i n p u t " a t l o w e r t e m p e r a -

t u r e s si n c e t h e b e n e f it s o f a n y s u c h s e c t i o n i n g

' h e a t ' a r e p r e s u m a b l y b e s t o w e d o n a l l

c r y o m i c r o t o m i s t s w i t h o u t d i sc r i m i n a ti o n

O n e c a n o f f e r t h r e e s p e c u l a t i o n s . F i r s t , t h a t

t h e l a r g e i c e c r y s t a l s i n t h e i r s p e c i m e n s

p r o m o t e d i s c o n t i n u o u s c h i p p i n g a t l o w

t e m p e r a t u r e s ( S a u b e r m a n n , I 9 8 0 ). S e c o n d ,

t h e v i c e ,t y p e h o l d e r t h e y U se ( S a u b e r m a n n ,

9 8 0 ) c a n n o t h o l d a fr o z e n s p e c i m e n w i t h o u t

p r o d u c i n g f ra c t u r e s i n t h e b ri t tl e b l o c k w h e n

i t is f i r m l y t i g h t e n e d ( se e a b o v e ) . T h i r d ,

t h e i r t h i n r a z o r - b l a d e k n i f e m a y v i b r a t e

u n d e r t h e w o r k - l o a d .

I n s u m m a r y , t h e s e c ti o n i n g s y s t e m d e -

s c ri b ed b y S a u b e r m a n n

et al.

( 1 9 8 1 a ) s e e m s

l es s s u i t a b le t h a n t h e a p p a r a t u s u s e d i n o t h e r

l a b o r a t o r i e s f o r c u t t i n g s m o o t h c r y o s e c t i o n s

b e l o w - - 3 0 C .

Ill Cryosectioning temperature for analysing

f r o z e n ' h y d r a t e d

s e c t i o n s

( i ) T h e r e a r e s e ve r a l r e a s o n s w h y i t is i m p o r t -

a n t t o c u t c r yo S e c ti o n s f o r m i c r o an a l y t ic a l

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628

w o r k a t a t e m p e r a t u r e a s l o w a s p o s s i b le

( G u p t a e t a L , 1 9 77 , 1 9 8 0 b ). A p a r t f l ' o m t h e

q u e s t i o n s o f c r y s t a l l i n e p h a s e t r a n s i t i o n s i n

f r o z e n t i s s u e - w a t e r , c a u s i n g l a r g e r i ce -

c r y s t a l f o r m a t i o n a b o v e - 8 0 C a n d o f

m o b i l i t y o f i o n s c a u s i n g r e d i s t r i b u t io n , t h e

c r y o c h a m b e r t e m p e r a t u r e h a s s e r i o u s i n -

f l u e n c e o n t h e r e t e n t i o n o f w a t e r b y th e c r y o -

s e c t io n s . T h e m a i n r e a s o n f o r a n a l y s i n g

f r o z e n - h y d r a t e d ( r a t h e r t h a n f r o z e n - d r i e d )

c r y o s e c t i o n s i s t h e p o s s i b i l i t y o f m e a s u r i n g

t h e c o m p o s i t i o n o f f r e e - f l u i d s p a c e s ~ u c h a s

t h e i n t e r c e l l u l a r s p a c e s , l u m e n s , ~ l a r g e v a c u -

o l e s . A t t e m p e r a t u r e s m u c h a b o v e - 7 0 C ,

i ce r e c r y s t a l l i z a t i o n a n d s u b l i m a t i o n p r o c e e d

r a p i d l y i n s u c h s p a c e s l a c k i n g a m a c r o m o l e -

c u l a r o r g a n i c m a t r i x , s o tl~ at t h e c o n t e n t s

G U P T A A N D H A L t ,

in s u c h c o m p a r t n l e n l s e i t h e r s h a t t e r a n d

d r o p o u t o n s e c t i o n i n g , o r e l s e a r e t o o d e -

h y d r a t e d f o r q u a n t i t a t i v e a n a l y s i s ( G e h r i n g

e t a L , 1 9 7 2; W . F u c h s , in D i s c u s s i o n a f t e r

G u p t a , 1 9 79 ; G u p t a a n d H a l l , 19 8 2) , A s

n ; e n t i o n e d b e f o r e , w e h a v e b e e n a b l e t o

a n a l y s e t h e c o n t e n t s o f f r e e - f l u i d s p a c e s i n

a v a r i e t y o f f l u i d ~

r a n s p o r t i n g e p i t h e l i a i n

c r y o s e c t i o n s c u l < ~ 6 0 C ( e . g. F i g s. - 8 ,

I 5 ) . W e r e g u l a r l y u s e

C a i l i p h o r a

s a l i v a r y

g l a n d s w i t h a l a r ge f r ee -f lu i d l u m e n o f k n o w n

i o n i c c o m p o s i t i o n t o c h e c k t h e ' r e li a bi li ty

o f o u r p r o c e d u r e s ( F ig . 1 5).

( it ) T o d e m o n s t r a t e t h a t t h e i r c r y o p r e -

p a r a t i v e t e c h n i q u e s d o n o t c a u s e r e d i s t r i-

b u t i o n o f e l e ct r ol y t es , S a u b e r m a n n e t a l

( 1 9 8 1 b ) h a v e a n a l y s e d a r t i f i c i a l g r a d i e n t s

F i gs . I - 6 . S c a n n i n g e l e c tr o n m i c r o s c o p e J X A - 5 O A ) i m a g e s t o s h o w t h e q u a l i ty o f

a p p r o x i m a t e l y I /~ m t hi c k e r y o s e c t i o n s o f C a l l ip h o ra s a l i v a r y g l a n d s c u t < - - 6 5 C .

F i gs . I - 3 a r e S T E M i m a g e s w i th p a r t i a l d a r k - f i e l d i l l u m i n a t i o n ; F i gs . 4 - 6 a r e c o r r e -

s p o n d i n g i m a g e s in t h e s e c o n d a r y o r r e f l ec t iv e m o d e . B e f o r e q u e n c h - f r e e z i n g t h e g l a n d s

w e r e o p e r a t i n g i n r i t ro i n n o r m a l D e x t r a n - R i n g e r ( R ) w i t h a n o s m o t a li t y o f a b o u t

3 20 m O s m o l . T h e c r y o s e c t io n s w e r e c u t . w i t h t h e c r y o c h a m b e r a n d t h e s p e ci m e n

t e m p e r a t u r e a t - - 6 9 C a n d t h e s te e l k ni f e a t - - 6 6 C . F i gs . 1 a n d 4 s h o w t h r e e s m o o t h

s e c t i o ns i n h y d r a t e d s t a t e . S e c t i o n a is t h i n n e r t h a n s e c t i o n s b a n d c . U p o n E P X M A ,

s e c t io n a w a s , f o u n d t o b e a b o u t 8 0 70 h y d r a t e d w h i l e s e c ti o n s b a n d c w e r e a l m o s t

f u l l y h y d r a t e d . A f i e ld f r o m s e c t i o n s a a n d b is s h o w n a t a h i g h e r m a g n i f i c a t i o n i n

F i g s . 2 a n d 5 . D c x t r a n - R i n g e r R ) h a s s e c t i o n e d s m o o t h l y . D u e t o s h e a r i n g e f f e c t s

d u r i n g c r y o s e c t i o n in g , t h e b o u n d a r y o f t h e c e l lu l a r e p it h e l iu m ( C e ) c a n b e r e c o g n i z e d

i n b o t h s e c t i o n s a n d i t is a l s o p o s s i b l e t o i d e n t i f y s e c r e t o r y c a n a l i c u l i , e s p e c i a l l y in t h e

p a r t i a ll y h y d r a t e d s e c t i on a . T h e f lu i d c o n t e n t s o f th e l u m e n ( L ) c o n t a i n i n g v i r t ua l l y

p u r e K C I s o lu t i o n ( a b o u t 1 65 r a M ) d o n o t c u t s m o o t h l y a n d a r e f r a g m e n t e d d u r i n g

s e c t i o n i n g , w h i c h h e l p s in t h e i d e n t if i c a t i o n o f l u m e n e v e n i n f u l l y h y d r a t e d s t a t e . E D S

s p e c t r a f r o m t h e D e x t r a n - R i n g e r a n d t h e l u m e n c o n t e n t s f r o m t h e s e s e c t i o n s a r e sh o w n

i n F i g . 1 5a . A f t e r t h e a n a l y s e s i n h y d r a t e d s t a t e t h e s e c t i o n s w e r e f u l l y d r i e d w i t h i n

t h e m i c r o a n a l y s e r c o l u m n a n d r e - a n a l y s e d. I m a g e s f r o m s u c h f r o z e n - d r i e d s e c t i on s a r e

s h o w n i n F i g s. 3 a n d 6 a n d c o r r e s p o n d t o F i g s . 2 a n d 5 . N o t e a v a s t i m p r o v e m e n t i n

c o n t r a s t , a c o m p l e t e l o s s o f c o n t e n t s f r o m t h e l u m e n ( L ) a n d t h e c a n a li c u li . A s t e r is k s

i n F i g . 3 s h o w t h e r a s t e r m a r k s l e ft b y t h e l a t e n t m a s s l o s s f r o m t h e a r e a s a n a l~ cs ed i n

t h e h y d r a t e d s t a t e. I n a d d i t i o n t o t h e i m a g e c h a n g e s a f t e r d r y i n g . t h e r e i s a l s o a g r e a t

i m p r o v e m e n t in t h e p e a k t o b a c k g r o u n d r a t i o s i n t h e X - r a y s p e c t r a f r o m s u c h f r o z e n -

d r i ed se c t io n s ( see F ig . 1 5 d , e ) . M ag n i f i ca t io n s : F ig s . 1 an d 4 , 3 0 0 ; F ig s , 2 , 3 , 5 , 6 ,

x 600

F i gs . 7 , 8 . A s a b o v e b u t f r o m g l a n d s o p e r a t i n g ht t , i lro i n a h y p o t o n i c 1 5 5 m O s m o I )

D e x t ra n : -R i n g e r, c r y o s e c ti o n e d a t t h e c h a m b e r a n d s p e c i m e n t e m p e r a t u r e o f - - 9 4 C

a n d k n i f e a t - - 9 2 C , T h e t i s s u e h a s c u t l e s s s m 0 o t h l y b u t t h e c o n t e n t s o f h i g h l y d i l at e d

l u m e n c o n t a i n i n g a b o u t 7:5 m M K C I a r e i n t a c t. T h e s e s e c t i o n s w e r e n e a r l y f ul ly

h y d r a t e d . I n t h e c e l l u l a r l a y e r ( C e ) o n e c a n r e c o g n iz e n u c le i ( a r r o w s ) a n d s e c r e t o r y

c a n a l J c u l i ( C n ) . T h e r e l a t i v e c o n t r a s t i n t h e i m a g e i s i n c r e a s e d b y ( 1) th e s l i g h t l y

d i f f e r e n t ia l d e h y d r a t i o n , ( i t) t h e s h e a r i n g e f f e c t s d u r i n g c r y o s e c t i o n i n g , ( ii i ) t h e u s e o f

p a r t i a l d a r k -f i e l d i l l u m i n a t i o n , a n d ( iv ) t he u s e o f e x t e n s i v e e l e c t r o n i c c o n t r o l s a v a i l a b l e

f o r p ro c e s s i n g t h e C R . T i m a g e . E D S s p e c t r a f r o m s u c h s e c t i o n s a r e s h o w n i n F i g .

1 5 b , c~ ' 2 5 0 0 :

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6 3 0

o f a m i x t u r e o f 15 ~/ o N a C I a n d 15 ~/o K I i n

b l o c k s o f 35 Y o g e l a t in e . T h e m e a s u r e m e n t s

w e r e m a d e i n c r y o s e c t i o n s o v e r i n t e r v a ls

o f 6 0 t~ m . H o w e v e r , a b l o c k o f 3 5 Yo g e l a t i n e

w i t h i t s e x c e l l e n t c r y o p r o t e c t i v e a n d w a t e r -

b i n d i n g p r o p e r t i e s w i t h u n i d i r e c t io n a l g r a -

d i e n t s p r o d u c e d b y 5 M p e r l i t r e o f s a l t

G U P T A A N D H A L L

s o l u t i o n c a n h a r d l y b e c o n s i d e r e d a r e a l i s t i c

m o d e l f o r s o f t b i o l o g i c a l t i s s ue s w i t h c o m p l e x

c o m p a r t m e n t s , a b o u t 2 0 ~ a v c r a g c d r y

w t a n d m u l t i d i r e c t i o n a I g r a d i e n t s o f d i f fe r e n t

i o n i c s p e c i e s i n l o w c o n c e n t r a t i o n s .

I n th e i r b i o l o g i c a l s t u d y t h e

demon

s t r a t i o n o f ' l i t t l e o r n o s m e a r i n g o r h e m o l y s i s '

F i gs . 9 - 1 4 . S c a n n i n g e l e c t r o n m i c r o s c o p e ( J X A - 5 0 A ) i m a g e s o f a p p r o x i m a t e l y

I / ti n th i c k c r y o s e c t i o n s c u t b e t w e e n - - 6 0 a n d - - 8 0 C f r o m a r a n g e o f ti s su e s to s h o w

t h a t t h e s e c t i o n s a r e s m o o t h a n d t h e m o r p h o l o g i c a l p r e s e r v a t i o n i s g o o d f o r t h e

p u r p o s e o f m i c r o p r o b e a n a l y s i s a t a r e s o lu t i o n o f < 0 - 2 5 /~ m .

F i g s. 9 , 1 0. S T E M i m a g e s f r o m a c r y o s e c t i o n o f a m e s e n t e r i c t e n t a c l e o f t h e s e a -

a n e m o n e

R/todactis

( L u b b o c k

et al.

1 98 1) i n f u l l y h y d r a t e d ( F i g . 9 ) a n d i n f u l l y d r i e d

s t a t e (F i g . 1 0) . T h e c r y o s e c t i o n w a s c u t a t a b o u t - - 6 5 ~ C . T h e D e x t r a n - c o n t a i n i n g

s e a - w a t e r ( S w ) a n d t h e n e m a t o c y s t c o n t e n t s ( N e ) w i th a b o u t 5 0 0 - 6 0 0 n lM o f C a h a v e

c u t s m o o t h l y b u t t h e o t h e r e c t o d e r m a l c e l ls ( C e ) w it ll t h e i r d e n s e m i n e r a l iz e d g r a n u l e s

e x h i b i t f l u t te r ( ' d i s c o n t i n u o u s c h i p p i n g ' ) , x 6 0 0 .

F i gs . 1 - 1 4 . S T E M i m a g e s o f c r y o s e c t i o n s a f t e r c o m p l e t e d e h y d r a t i o n i n t h e S E M .

F i g . l I . A c e l l f r o m Calliphora s a l i v a r y g l a n d w i t h t h e b a s a l l a m i n a ( B l ) , c a n a l i c u l i

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

p r o b a b l y m i t o c h o n d r i a ( se e G u p t a et aL 1978a) . 7000 .

F i g. 1 2. T h e c r y p t a r e a o f th e m u c o s a i l a y e r o f t h e r a b b i t i l e u m , q u e n c h - f r o z e n a s a

1 c m d i a m e t e r s h e e t a f t e r o p e r a t i n g in ritro i n a n U s s i n g - t y p e c h a m b e r f o r 1 h ( G u p t a

etaL 1 9 7 8 b ; G u p t a a n d H a l l , 1 9 78 , 1 9 79 , 1 98 1 a ). N o t e t h e b r u s h - b o r d e r ( B B ) , l a m i n a

p r o p r i a ( L p ) a n d n u c l e i i n s e v e r a l c e ll s . 1 60 0.

F i g. 1 3. F r o m a r a c e m o s e s a l i v a r y g l a n d o f t h e c o c k r o a c h Perip/aneta americams

s h o w i n g t h e f l u i d - s e c r e t i n g ' p e r i p h e r a l c e l l s ' ( P ) , a c i n i o f s e r o u s c e l l s w i t h d e n s e s e c r e -

t i o n g r a n u le s a n d l u m i n a ( L ) , a n d t h e s u r r o u n d i n g D e x t r a n - R i n g e r ( R ) . 2 3 00 .

F i g . 1 4 . A s i n F i g . 1 3 , b u t s h o w i n g t h e n u c l e u s ( N ) a n d t h e m i c r o v i l l i ( M y ) i n a

p e r ip h e ra l ce ll . 1 3 ,0 0 0 .

F i g . 15 . X - r a y e n e r g y s p e c t r a ( E S D ) b e t w e e n 0 .8 a n d 5 .2 k e V f r o m / Lm t h i c k

c r y o s e c t i o n s o f Calliphora s a l iv a r y g l a n d s w h ic h , w h e n b a t h e d i n s o d i u m - R i n g e r

( + D e x t r a n ) , s e c r e t e a n i s o t o n i c K C I f l u i d ( G u p t a et aL 1 9 7 8 a : G u p t a a n d H a l l , 1 9 7 9 ,

1 98 1 a) . A ll s p e c t r a w e r e a c q u i r e d w i t h a b e a m o f 45 k V a n d 1 .0 n A p r o b e c u r r e n t o v e r

1 0 0 s ec l i v e - t im e ; t h e y a r e a l l d i s p la y e d a t t h e s a m e s c a l e . T h e c h a r a c t e r i s t i c p e a k s

f r o m a l u m i n i u m ( A l ) a r e f r o m t h e s u b s t r a t e fi lm a n d t h e D u r a l i u m h o l d e r ; t h e r e a r e

n o o t h e r e x t r a n e o u s p e a k s i n t hi s e n e r g y b a n d . ( a) C o m p a r i s o n o f D e x t r a n - R i n g e r

( N a , 13 5 m M ; C I , 1 42 r a M ; K , I 0 m M ; C a , 2 r a M ; 3 20 m O s m o l ; b a r s ) w i t h t he

l u m e n c o n t e n t s ( C l , 1 65 m M ; K , I 6 0 m M : d o t s ) in a f u ll y h y d r a t e d s e c t i o n ( c f . F i g . 2 b ) .

( b , c ) F r o m a h y d r a t e d s e c t i o n o f a g l a n d f r o z e n w h i l e o p e r a t i n g in eitro i n a n h y p o t o n i c

( 1 5 5 m O s m o l ) D e x t r a n - R i n g e r ( N a , 5 0 r a M ; C l , 5 6 m M ; K , 1 0 r a M ; C a , 2 r a M ) .

( b ) C o m p a r i s o n o f D e x t r a n - R i n g e r ( b a r s ) w i th t h e l u m e n c o n t e n t s ( C I, 70 r a M ; K ,

7 5 r a M ; d o t s ) ; ( c ) c o m p a r i s o n o f a n u c le u s ( b a r s ) w i t h t h e l u m e n c o n t e n t s ( d o t s ) .

N o t e t h a t t h e c o n t i n u u m l e ve l s a r e v i r t u a l ly t h e s a m e i n F i g. 1 5 a - c i n d i c a t in g t h e u n i -

f o r m i t y i n t h i c k n e s s a n d l e v e l o f h y d r a t i o n o f th e s e c r yo s e c f i o n s . ( d ) F r o m t h e s a m e

c r y o s e c t i o n s a s i n F i g . 1 5 a , b u t a f t e r c o m p l e t e d e h y d r a t i o n i n t h e S E M ( c f . F ig . 3 ) t o

s h o w t h e c o m p a r i s o n o f D e x t r a n - R i n g e r ( b a rs ) w i th t h e c i r c u m n u c l e a r c y t o p la s m

( d o t s ). T h e l u m e n c o n t e n t s a r e l o s t in s u c h d e h y d r a t e d s e c t i o n s . (e ) F r o m a c r y o s e c t i o n

i n F i gs . 1 - 3 t o s h o w t h e c h a n g e i n c h a r a c t e r i s t i c p e a k / ' c o n t i n u u m r a t i o s o f D e x t r a n -

R i n g e r s p e c t r a f r o m a f u l l y h y d r a t e d ( d o t s ) t o a f u l ly d e h y d r a t e d s t a t e ( b a r s) . T o a v o i d

t h e e f f e ct o f l a t e n t m a s s l o s s t h e a r e a s a n ~ y s e d i n t h e h y d r a t e d s t a t e ( a s t e r i s k s i n F i g .

3 ) w e r e n o t u s e d f o r a n a l y s e s i n t h e d e h y d r a t e d s e c t i o n . I n t h is ' i d e a l" e x a m p l e t h e o n l y

s i g n if i c a nt c h a n g e i n t h e s p e c t r a i s t h e l o ss o f c o n t i n u u m d u e t o r e m o v a l o f H 2 0 .

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M1CROANALY SIS Ol FROZEN-H YDRATED SI :DI~CIMENS

633

(Fig. 3F in Buiger ( , t a L , 1981) is not very

sound. The marker for an iron peak has

been mis plac ed: it is shown near 5.9 keV,

the quantum energy of the m tlng , ctsles(,

radiati on e mitted by ZZFe, the radi oactive

isotope used by most detector manufactu rers

for the test data delivered to their customers.

The m ark er should be at 6.4 keV, where there

is indeed a suggestion of an iron peak, as

much as one would expect front haemolysis

since a red blood cell itself gives only a small

Fe peak. The huge potassium peak in this

spectrum fi'om capillary plasma (especially

the extraordinary K/Na ratio) can only be

understood either on the basis of haemolysis

or a gross redistribution of potassium front

some other compartments, or both. The high

potassium peak in Fig. 4E from the collecting

duct lumen suggests the same. These anoma-

lies in the spectra front the collecting duct

lumen and capillary plasm ma y be the reason

why quantitative data front these compart-

ments are not given in the results (Table 11

in Bulger e t a L , 198t).

IV. Specimen support, transfer and main-

tcnance in the SEM

The second major claim of Saubermann

e t a L (1981a, p. 261) is that they have now

met a previously unsatisfied need in the

handling of N'ozen-hydrated sections: there

was no simple system for specimen support,

transfer, and maintenanc e at low tempera-

ture within the instrument (10)'. The latter

reference is to a review by Hall (1979a),

but the re is no such statement there. Possibly,

Saubermann e t a L had in mind the comment

on p. 151 (Hall, 1979a): The study of frozen-

hydrated sections is in principle the least

objectionable of the alternatives, though

maintenance of hydration is proving difficult

in practice .. . Hydration in this context

implied totally unimpaired hydration of

the section, including matrix-free fluid

spaces, even during the analysis of very

small reg ions ( < 0.1 tzm-). Thi s is a realisti c

note of caution. In the articles by Sauber-

mann

e t t t l .

(1981a, b)the genuine elemental

analysis in fro zen-hy drated sections is

confined to the study of saline standards

containing 20~ or more organic polymers;

all ele men tal analyses from the biological

specimens were obtained front j ? o z e n - d r i e d

sections. Nowh ere have t he y presented an

analys is o f a ma t r ix -f i ee f lu id space, let

a lone nar row channe ls requ i r ing a s ta t i c

probe o f about 0 .1 I l l l s (Gupta e/ a/ . ,

1978a, b, 1980 b~ Gu pta and Ha l l , 1979~

1981a). Apart front th e sectioning tempera-

ture discussed above, the specimen support

and maintenance system of Saubermann

e t t t l . may be inadequate for such analyses.

Si ; e ( i me t t SUl ; l;or l

(i) i n ou r procedure cryosections are mounted

on aluminized nylon lilms stretched over

2-3 mm hole in a Du raliu m collar. The

rim of the hole is carefully machined to

give a kn ife-edge and the use of a metal

grid has been avoided in order to reduce

the contribution from extraneous sources

to X-ray continuum (Gupta e t a l . , 1977;

Hall, 1979b). The bars in mesh -type EM

grids also block a large proportion of the

sections and therefore prevent the analyses

of the same areas in all the serial sections on

a holder (cf. Rick e t a l . , 1979); such analyses

are often desirable in order to assess the

level of hydration retained and to reduce

statistical errors. The holders are 'flashed'

just before use in a sputter-coater to remove

hydrophobicity and the sections are gently

pressed dow n with a polished copp er th umpe r

to promote good contact. As noted above

aluminized nylon flints do provide an ex-

cellent substrate for analysing hydrated

sections but the high alu min ium K0 X-rays

front the substrate film and the holder in our

X-ray energ y spectra (Fig. t5) have been

a problem, especially for EDS measurements

of Na and Mg but not for WDS (Hall and

Gupt a, 1982). We are no w experinaenting

with multislot nickel grids and carbon,

nickel or chromium coating (Marshall,

1980) on different metal holders with some

success. When positioned in the SEM the

holders are in direct contact with the cold

stage of the microanalyser (Gupta e t a l . ,

1977; Gupta and Hall 1979, 1982; Taylor

and Burgess, 1977).

(ii) Sauberma nn e t a L (1981 a) use the same

type of knife-edge holder as ours but made o f

beryllium. A beryUium grid is placed on the

hole and covere d with a nylon film. The cry o-

sections are directly placed on the carb on-

coated nylon film. The holder is then placed

into a groove d copper t ransp orte r which in

turn is slid into the cold stage. Such a speci,

men mounting arrangemen t will undoubt edly

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6 4

p r o v i d e a s u b s t a n t i a l r e d u c t i o n i n th e ex -

t r a n e o u s c o n t i n u u m b a c k g r o u n d a n d g e n e r -

a t e cl e a n e r E D S s p e c t ra . T h e s p e c i m e n

s t a b i l it y i s c l a i m e d t o b e g o o d u n d e r v e r y

l o w b e a m c u rr e n t s ( m a x i m u m a b o u t 0 .I n A

i n 1 t ~m -) . I t i s n o t s t a t e d h o w t h e s e c t i o n s

w i ll b e h a v e t r a d e r h i g h e r c u r r e n t d e n s i t i es

n e c e s s ar y f o r f in e r r e s o l u t i o n a n d f o r W D S

n a e a s u r e m e n t s n e c e s s a r y f o r a n a l y s i n g l o w

l ev e ls o f i n t r a c e ll u l a r N a a n d C a d i r e c t l y

i n h y d r a t e d

s e c t i o n s ( s e e b e l o w ) .

Spe ein len It t lt lsporl

(i) i n o u r m e t h o d t h e h o l d e r s b e a r i n g f r o z e n -

h y d r a t e d s e c ti o n s a re i m m e d i a t e l y t ra n s -

f e rr e d f r o m t h e c r y o s t a t t o a s t o r a g e D e w a r

u s i n g a p r e - c o o l e d s l i d i n g r o d a n d s h e a t h

d e v i c e ( H u t c h i n s o n

e t a L

1974, 1978;

G u p t a , 1 9 76 ; S a u b e r n a a n n a n d E c h l in

1 97 5 ; G u p t a

e t t tL

1 9 7 7 ) . T h e s t o r a g e D e w a r

c o n s i s t s o f a l a r g e m e t a l b l o c k , w i t h s h a p e d

s l o t s f o r s e v e r a l s e c t i o n h o l d e r s , h o u s e d i n

a 5 l it re L N o _ D e w a r ( U n i o n - C a r b i d e ) .

T h e s e c t io n h o l d e r s i n t h e sl o t s a r e p r o -

t e c te d b y a r o t a t i n g s h u t t e r a n d r e m a i n a t

< - 1 9 0 C w i t h o u t d i r e c t c o n t a c t w i t h L N e :

t h e s e c ti o n s r e m a i n q u i t e s t a b l e w i t h o u t l o s s

o r g a i n o f m o i s t u r e f o r w e e k s w i t h r e p le n i s h -

m e n t o f L N ._ , e v e r y 2 d a y s ( G u p t a

e t a L

1 9 7 7 ; G u p t a a n d H a l l , 1 9 8 2 ) . F o r m i c r o -

a n a ly s i s in t h e J X A - 5 0 A t h e s e c ti o n h o l d e r s

a r e t r a n s f er r e d f r o m t h e s t o r a g e D e w a r

t h r o u g h t h e a i r -l o c k i n t o t h e m i c r o a n a l y s e r

b y . u s i n g a p r e - c o o l e d f l a n g e d d e v ic e w i t h a n

i n s u la t ed c o p p e r c a p ( G u p t a

e t a L

1 9 7 7 ;

G u p t a , 1 9 7 9 ; G u p t a a n d H a l l , 1 97 9 , 1 9 82 ).

I n t h i s a r r a n g e m e n t t h e p r o c e s s es o f c r y o -

s e c t i o n i n g a n d m i c r o a n a l y s i s c a n p r o c e e d

i n d e p e n d e n t l y .

( i i ) S a u b e r m a n n

e t a L

( 1 9 7 7 b ) i n t r o d u c e d

a c o m p l i c a t e d ' s a t e l l i t e ' s y s t e m f o r t r a n s ,

p o r t i n g h y d r a t e d c r y o s e ct i o n s

i n vaq t t o

i n t o

t he S E M , N o w t h ey h h ve a d o p t e d o u r s i m p l e

a n d p r a c ti c a l a p p r o a c h o f a f la n g e d d e v i c e

w i t h a p r o t e c t i v e c a p f o r t h i s p u r p o s e

( S a u b e r m a n n

e t a L

I 9 8 1 a ) . H o w e v e r , : i n

t h e i r s y s t e m t h e r e i s n o p r o v i s i o n f o r s t o r i n g

t h e s e c t i o n h o l d er s . O n c e t h e c r y o s e c t i o n s

h a v e b e e n n a o u n t e d o n a h o l d e r i n t h e m i c r o -

t o m e it m u s t b e l o a d e d o n tO t ae m i c r o s c o p e

c o l d , s t a g e i m m e d i a t e l y ... D e t a i l e d a n a ly s i s

o f g o o d : f r o z en - h y d r a t ed s e c ti o n s c a n t a k e

s e v e r a l / h o u r s . I n : t h e m e a n t i m e t h e f r o z e n

t i s s u e b l o c k e i t la e r h a s t o r e m a i n i ll t h e c r y o -

m i c r o t o m e a t - 3 0 C a n d b eg in t o d r y , o r

G U P T A A N D H A L L

t h e c r y o c h a m b e r is c o o l e d to - 8 0 C a n d

t h e y r e - eq u i l i b ra t e d to - 3 0 C w h e n f u r t h e r

s e c t i o n s a r e r e q u i r e d . B o t h t h e a l t e r n a t i v e s

a r e l i k e l y t o b e h i g h l y d e l e t e r i o u s t o t h e

s p e c i m e n .

C o l d s t a g e

( i) T h e L N ,_ , c o o l e d c o l d - s t a g e i n t i l e J X A -

5 0 A w a s b u i l t in ou t" l a b o r a t o r y ( T a y l o r a n d

B u r g e s s, 1 97 7 ), (a ) t o a l lo w s i m u l t a n e o u s

s c a n n i n g t ra n s m i s s io n a n d o t h e r m o d e s o f

i m a g e f o r m a t i o n , ( b ) t o m a i n t a i n a t e m p e r a -

t u r e o f < - 1 7 0 C w i t h t h e s p e c i m e n p r o b -

a b l y I 0 - 2 0 C w a r m e r , ( c ) t o h a v e a n e f f i ci e n t

L 'N -. c o o l e d a n t i c o n t a m i n a t i o n p l a t e p o s i -

t i o n ed a b o u t 1 m m a b o v e t h e s p e c i m e n a n d

m a i n t a i n e d a t < - 1 9 0 C , ( d) t o h a v e a

r e a d i l y a c c e s s ib l e F a r a d a y c a p t o r e g u l a r l y

m e a s u r e t h e b e a m c u r r e n t , (e ) to h o u s e

s e v e r a l s t a n d a r d s f o r c h e c k i n g t h e p e t ' f o r -

m a n c e o f s p e ct r o m e t e rs a n d f o cu s t h e W D S ,

( f ) t o h a v e h e a t i n g f a c i l i t i e s f o r w a r m i n g

t h e s ta g e t o d e h y d r a t e t h e s e c ti o n s . S a u b e r -

m a n n ( 1 9 7 9, p p . 6 1 1 - 6 1 2 ) h a s q u i t e e r r o n -

e o u s l y s t a t e d t h a t o u r co l d s ta g e ' . . . c a n n o t

b e u s e d fo r S T E M i m a g i n g ' .

( i i ) S a u b e r m a n n

e t a l .

( 1 9 7 7 b , 1 9 8 1 a ) h a v e

b u il t a c o l d s ta g e s i m i l ar t o o u r s w i t h S T E M

i m a g i n g f a c i l i t i e s b u t w i t h o u t a n y s e p a r a t e

a n t i c o n t a m i n a t i o n d e v i c e . I n t h e i r s y s t e m

t h e S E M c o l d - s t a g e i t s e lf a c t s a s a c o n -

t a m i n a t i o n t r a p a n d i s b e l i e v ed t o b e 1 0 -

1 5 C c o l d e r t h a n t h e s p e c im e n . O n t h e o r e ti c a l

g r o u n d s a n d i n o u r e x p e r i e n c e t h is i s n o t

s u f f ic i e n t : t h e r e is n o r ea l b a r r i e r t o c o n t a m i -

n a n t m o l e c u le s in t h e c o l u m n s t r i k in g t h e

s p e c i m e n a n d f r e e z i n g t h e re . T h i s s y s t e m

i s e s p e c i a l l y i n a d e q u a t e w h e n t h e s t a g e i s

w a r m e d t i p t o d e h y d r a t e t h e s p e c i m e n f o r

a n a l y s is : t h e r e is a h i g h r i sk o f c o n t a m i n a t i o n

e s p e c i a l l y f r o m C I a n d S b y s e l e c ti v e a d s o r p -

t i o n i n c e rt a i n t i ss u e c o m p a r t m e n t s ( H a ll

a n d G u p t a , 1 9 8 2 ) .

V. Ima g ing o f se c t io ns in S E M mic r o -

analysers

I n o r d e r t o p e r f o r m X - r a y m i c r o a n a l y s i s

o n e , fi rs t i d e n t if i e s c e ll a n d t i s s u e c o m p a r t -

m e n t s i n a n i m a g e o f th e s p e c im e n a n d t h e n

l o c a te s a f o c u s e d b e a m o f s u i ta b l e s iz e w i t h i n

t h e i m a g e . A s m o o t h c r yo s e ct io n b y d e -

f i n it i on ~ h a s n o s u r f a c e c o n t o u r s a n d h e n c e

pr o du c e s a f e a tur e l e ss se c o nda r y e l e c tr o n

ima g e e .g . F ig s . 1 - 8 ) . Ba c a ne r

e t a l

1973)

8/10/2019 Artigo Mev

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M I CROANAL YSI S OF Fr ~OZ E N I I YDRAT

i n t r o d u c e d t h e u s e o f si l ic o n p h o t o c e l l

d e t e c t o r f i t t e d u n d e r a h o l e i n t h e c o l d - s t a g e

o f a C a m b r i d g e - S 2 S E M t o o b t ai n S T E M

i m a g e s o f t h i n ' f i 'o z e n - h y d r a t e d ' c r y o s e c t i o n s .

T h e s a m e a p p r o a c h h a s be e n a d o p t e d b y

M o r e t o n

e t a L

( 1 9 7 4 ) a n d S a u b e r m a n n

e t a L

( 1 97 7 , 1 9 81 a ). T h e J E O L J X A - 5 0 A

u s e d b y u s a n d t h e C a m b r i d g e S -1 8 0 S E M

( R o s s

e t a L ,

1 9 81 ) a r e f i tt e d w i t h a p h o s p h o r -

t i p p e d l i g h t- p i p e a n d a p h o t o n m l t i p l i e r

u n d e r t h e c o l d s t a g e t o p e r m i t S T E M i m a g -

i n g . T h e l a t t e r a r r a n g e m e n t i s m o r e s e n s i t i v e

a n d a l s o h a s e x t e n s i v e f a ci li ti e s f o r e l e c t r o n i c

p r o c e s s i n g o f t h e i m a g e s . O t h e r c o m m e r c i a l

s y s t e m s a r e n o w a v a i l a b l e w i t h h i g h r e s o l u -

t i o n S T E M f a c il it i es f i tt e d t o 1 0 0 - 2 0 0 k V

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 s p r o -

d u c e d b y v a r i o u s m a n u f a c t u r e r s ( e. g. Z i e r o l d ,

1982) .

I m a g e q l ta l iO i tt h y d r a t e d c t g o s e c t i o n s

( i ) I t w a s n o t e d b y M o r e t o n e t a l . ( 1974 )

a n d b y G u p t a a n d H a l l ( s ee G u p t a , 1 9 76 )

t h a t a l t tn a t h i c k f u l l y h y d , ' a t e d s e c t i o n

d o e s n o t u s u a l l y r e v e a l a n y m o r p h o l o g i c a l

d e t a i l e i t h e r i n t h e s e c o n d a r y o r S T E M

i m a g es . W h e n t h e se c t io n s w e r e d e h y d r a t e d

w i t h in t h e S E M c o l u n m t h e re w a s a d r a m a t i c

i m p r o v e m e n t in t h e c o n t r a s t a n d d e ta i ls

i n t h e i m a g e s . I n s u b s e q u e n t s t u d i e s G u p t a

e t a L

( 1976 , 1977 , 1978a , b , 1980a , b ) f ound

t h a t a c a r e f u l a n d s l i g h t d e h y d r a t i o n o f t h e

s e c t io n s ( 1 0 - 2 0 % l os s o f H ,, O ) p r o d u c e d

s u f fi c ie n t d e t a i l s in S T E M i m a g e s s o t h a t

t h e m a j o r i n t ra - a n d e x t r a ce l l u la r c o m -

p a r t m e n t s e v e n i n c o m p l e x t i ss u e s c o u l d b e

r e c o g n i ze d a n d m e a n i n g f u l l y a n a ly s e d . T h e

u s e o f a m i x e d b r i g h t a n d d a r k - fi e l d i m a g i n g

f u r t h e r i m p r o v e d t h e c o n t r a s t . T h e s e o b s e r -

v a t i o n s o n i m a g e d e t ai ls h a v e n o w b e e n

c o n f i r m e d i n s e v e r a l o t h e r l a b o r a t o r i e s

u s i n g 1 00 n m t h i c k c r y o s e c t i o n s i n a 3 0 k V

S E M ( R o s s e t a L , 1 9 8 1 ) , i n a n A E I h i g h

v o l t a g e E M ( H u t c h i n s o n

e t a L ,

1 9 7 8 ) a n d

i n a n E l m i s k o p S T 1 00 F S T E M w i t h a

f i el d e m i s s i o n g u n ( Z i e r o l d , 1 9 8 2 ). R e -

c e n t l y , h o w e v e r , w e h a v e n o t e d t h a t e v e n

i n f u l l y h y d r a t e d s e c t i o n s i t i s p o s s i b l e t o

i d e n ti f y f e a t u r e s w h i c h h a v e d i s p r o p o r -

t i o n a t el y h i g h c o n t e n t s o f e le c t r o l y te e l e m e n t s

s u c h a s C a , Z n , M g , C u , e tc . , a s c o m p a r e d

t o t h e s u r r o u n d i n g t i s s u e ( L u b b o c k

e t a L ,

198 I , F ig . 9 ) .

( i i ) I n t h e i r ' f i r m a n d w e l l t e s t e d ' m e t h o d s

S a u b e r m a n n a n d E c h l i n ( 1 9 7 5 ) p u b l i s h e d

ED SPECI MENS 635

h i g h l y d e t a il e d S T E M i m a g e s f r o m I r m a

t h i c k ' f u l l y h y d r a t e d ' s e c t i o n s o f s e v er a l

t i s s u e s a n d d i d n o t f i n d a n y c h a n g e i n t h e s e

i m a g e s w h e n t h e s e c t i o n s w e r e c o m p l e t e l y

d r i e d b y w a r m i n g t h e . s t a g e i n t h e C a m -

b r i d g e $ 4 S E M : t h e s t a g e c o n s t r u c t e d a n d

u s e d p r e v i o u s ly by M o r e t o n e t a l . ( 1974 ) .

U n t i l r e c en t l y S a u b e r m a n n h a s m a i n t a i n e d

t h a t t h e p r e s e n c e o f i m a g e d e t a i l s i s n o t a n

i n d i c a t io n o f d e h y d r a t i o n i n c r y o s e c ti o n s

f i 'o m s o f t t i ss u e s (s e e D i s c u s s i o n a f t e r G u p t a ,

1 9 7 9 ; S a u b e r m a n n , 1 9 7 9 ) . S a u b e r m a n n

e t a L

( 1 9 8 1a ) n o w c l a i m t h a t t h i n n e r (0 - 5 / t i n )

f u l ly h y d r a t e d s e c t i o n s f r o m t h e r a t r e n a l

p a p i ll a s h o w e d m o r p h o l o g y t h a t w a s n o t

v i s i b l e i n e q u a l l y h y d r a t e d t h i c k e r ( I i m a )

s e c t i o n s , a l t h o u g h t h e y n o w a c c e p t t h a t t h e

i m a g e d et a il s a r e t r e m e n d o u s l y e n h a n c e d

a f t e r t h e s e c t i o n s h a v e b e e n d r i e d , i t w o u l d

s e e m t h a t t h e p r e s e n c e o f i m a g e d e t a i l s

s u t' fi c ie n t t o r e c o g n i z e c o m p a r t m e n t s i n

r e l a t i v e l y t h i c k ( 0 . 5 / t i n )

f i d l y h y d r a t e d

s e c t i o n s o f s o f t t i ss u e s , e x a m i n e d i n b r i g h t -

f ie l d S T E M i m a g e s w i t h a 3 0 k V b e a m , i s a n

e x p e ri e n ce u n i q u e t o S a u b e r m a n n a n d h i s

c o l l e a g u e s . I t s h o u l d b e e m p l a a s i z e d h o w e v e r

t h a t i n t h e a b s e n c e o f a p e r i p h e r a l s t a n d a r d

( s e e b e l o w ) o r o t h e r s u i t a b l e c r i t e r i a ( D o w

e t a L ,

1 9 8 1 ; H a l l a n d G u p t a , 1 9 8 2 ) i t i s

n o t p o s s i b l e f o r S a u b e r m a n n

e t a l .

t o b e

s u r e t h a t t h e i r s e c t i o n s f r o m b i o l o g i c a l

t i s s u e s w e r e i n d e e d

f t d l y I t j d r a t e d

( a l so c f .

R o s s

e t a l . ,

1 9 8 1 ) . F u r t h e r , i t m u s t b e n o t e d

t h a t i n b o t h t h e i r

b i o l o g i c a l

s t u d i e s ( B u l g e r

e t a L ,

1 9 8 1 ; S a u b e r m a n n a n d R i l e y , 1 9 8 0 )

i t w a s n o t p o s s i b l e t o o b s e r v e a d e q u a t e

i m a g e d e ta i l s in h y d r a t e d s e c t i o n s a n d a ll

t h e e l e m e n t a l a n a l y s e s w e r e c a r r i e d o u t i n

f i d l y d r i e d

s e c t i o n s .

VI An alyt ical procedures

X - r a y s p e c t ro m e t r y

( i) O u r S E M m i c r o a n a l y s e r is f i tt e d w i t h t w o

f ul ly f o c u s in g W D S a n d o n e K e v e x - L i n k

E D S ( h o r i z o n t a l s p e c i m e n , t a k e - o f f a n g l es

3 5 , E D S d e t e c t o r a r e a 3 0 m m 2 a n d r e s o l u -

t i o n 1 55 e V F W H M ) . T h e X - r a y e n e r g y

s p e c t ra a r c d e c o n v o l u t e d w i t h a n o n - l i n e

c o m p u t e r u s i n g t i le L i n k S y s t e m s Q U A N -

T E M / F L S s o f t w a r e p r o g r a m d e v e lo p e d by

o n e o f u s ( T . A . H . ) i n ' c o l l a b o r a t i o n w i t h

D r P e t er S t a tl ? am o f L i n k S y s t e m s . T h e

p r o g r a m is b a s ed o n c o n t i n u u m n o r m a l i z a 7

t i o n p r o c e d u r e s ( H a l l, 1 9 71 ; G u P t a I e t

al . ,

8/10/2019 Artigo Mev

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6 3 6

1977) and includes a standards file, G-factor

iteration, mass-thickness standard, absorp-

tion corrections for characteristic X-rays

and a co'mprehensive subtraction fi'om the

continuum of background X-rays from

the substrate film as well as other e xtraneo us

sources such as the section holder, grid, etc.

All the relevant information together with

the elemental concentrations in mM per

kg (hydrated or d ry mass as the case

may be) with probab le errors is printed out

within abo ut 2-3 rain of every analytical

measurement or 'run'. The basic lay-out

of the instrume ntation with an example

of the print-out from an actual run on a

fully frozen-hydrated section is given in

another article (Gupta and Hall, 1982).

In fully hydrated sections using EDS and

the current deconvolution program one can

discriminate between Ca levels below 10 mM

in t he presence o f nor mal cell K ( .~ 100 mM

or mo re); the cut-off for Na, set by funda-

mental physical factors, is at 20-30ram

(Hall and Gupta, 1982, and in preparation).

With WDS we have measured Na concen-

trations below 10 mM arid Ca (as well as

oth er ions) down to mM directly in frozen-

hydra ted sections.

(ii) Saubermann

et aL

(1981a, b) have used

an AMR 900 SEM (AMRay, Burlington,

Mass.) operating at 30kV fitted with a

KEV EX model 5100 EDS (10 mtn - detec tor,

35 take-ofrangle, resolution 155eV F WHM )

interfaced to a min icom pute r (PDP I1/03).

The deconvolution was carried out using

a 'top ha t' digital band-pass filter (Statham ,

1976). The data reduction is based on Hall's

contin uum normalization and performed with

a simple interactive Fortran IV program.

No infor matio n is given On the time required

or on the nature of the information printed

o t i t It is claimed that apart from a small

contribution from the subs trat e film no

other X-ray background from extraneous

sou rce s is present. It is difficult to see why

there should be no continuum X-rays from

the beryllium grid and the holder, generated

by electrons s cat ter ed by a 0.5-1 /am thick

hydrated section ir radiate d with a relatively

low en er gy 30 kV beam, an d if there are such

X, rays h ow do es on e identify them? Char-

acteristic X-ray peaks from beryllium (energy

10 eV) ca nn ot be identified in EDS spectra.

Accor ding t011 the data given by Sa ub er -

mann et aL (1981. p, 270) their X-ray spectro -

G U P T A A N D

H A L L

metry is clearly inadequate for the study of

Na and Ca in frozen- hydrat ed sections of

normal cells. The deconvolution system

cann ot de,-il with < 20 mM of Ca in the pre-

sence of > 100 mM of K, and cann ot deal

with less than abou t 100 mM of Na (see their

Fig. 2). The slope for CI in Fig. 2 is approxi-

mately hal f of that for K and S. Since the

three elements are close in atomic number,

and equally wide X-ray energy bands were

used for them, the EDS X-ray sensitivities

for them must be nearly equal. The only

apparent explanation for the low CI slope

is loss of chlorine from the section under

the electron beam, suggesting less stability

of their specimens than is claimed.

z ~ 4 e a s t t re m e n t o . / a q t t e o t t s fi t c l i o n

(i) There are two basic theoretical formula-

tions which can be used to estimate per-

centage weight fi'action of wat er in specimen

areas analysed (Gupta et aL 1977). These

are:

'Method

A '

water = [ I - ( l'Vcleh.vd,'ate(J/

l~I /h. , ,,~, ., ,. ,,1) x I0 0 ( I )

M e t h o d B '

w a t e r =

[1

_ - [

h,~IW)

hydrated ]

C ~ a ~ ~ d J x

OO

2 )

mM

x

(kg wet wt)x-1]

mM x (kg dry wt)x-lJ

x Joo 3)

N refers to ~he cha,'acteristic X-ray counts

of an eleme nt x and t'V to the fully correc ted

continuum X-ray count s, both from th e

sam e analysed subarea of the specimen.

Equ ati ons (2) and (3) are essentially th e

same because ~in the conti nuum normaliza-

tion proced ure of data reductio n time ratio

of

N x / W

(R-va lue in Sauberrnann

et al.

1981b) from tile specimen being analysed is

multiplied by the conversion factor from

the st an da rd to arrive at the concentration

of t he element x in mM kg -1. 'Met hod A"

is subject to many experimental errors due

to shrinkage of sections upon drying, drift

in beam current, etc., whi le 'M et ho d B' is

relatively unaffected by such variations. We

have published experimental data from

biological specime ns illustrating th e advant-

ages of usin g 'Method B' (Gupta 1979;

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638

hh, ntification o f compartmentx by marker

elemetlts

( i ) S i n c e a p o s i t i v e v i s u a l i d e n t i f i c a t i o n o f

f i n e r c e l l a n d t i s s u e c o m p a r t t a a e n t s in h y d r a -

t e d c r y o s e c t i o n s i s n o t c e r t a i n , i t i s q u i t e

p r a c t i c al t o t a k e n o t e o f th e c h a r a c t e r i s t i c

X - r a y s i g n a ls f r o m a " m a r k e r ' c l e m e n t d u r i n g

o r a f t e r t h e a n a l y s i s t o c o n f i r m s u c h i d e n t if i -

3 .

c a t~ o n . F o r e x a m p l e , t o a n a l y s e n a r r o w e x t r a -

c e l l u l a r sp a c e s b y m e a n s o f a static probe o r a

fine raster w e h a v e o f t e n u s ed v er y l o w p h o s -

p h o r u s c o u n t s a s c o n f i rm a t i o n " t h a t t h e a d -

j a c e n t c y t o p l a s m h a s b e e n e x c l u d e d f r o m t h e

a n a l y s i s ( G u p t a a n d H a l l, 1 97 9). S u c h a

p h o s p h o r u s s i g n al m a y e v e n b e u s e d q u a n t i -

t iv e l y to e s t i m a t e a c o r r e c t i o n f o r t il e c o n t r i -

b u t i o n o f m i n u t e c y to p l a s m i c f ol d s , n o t

v i s u a l l y r e s o l v e d , t o t h e o b s e r v e d e x t r a c e i l u -

l a r s i g n a l s ( G u p t a a n d H a l l, 1 9 81 a) .

I n c a s e s w h e r e e x c e p t i o n a l l y h i g h c o n -

c e n t r a t i o n s o f s o m e e l e m e n t a r e p r e s e n t i n

c e rt a in c o m p a r t m e n t s (e .g . 5 0 0 -6 0 0 m M C a

i n t h e n e m a t o c y s t s ; L u b b o c k et aL, 981) i t i s

e v e n p o s s i b l e t o u s e X - r a y m a p s a s a n a i d

t o t h e p r e s e n t a t i o n o f d a t a .

( i i ) S a u b e r m a n n et aL ( 1 9 8 1 a , p . 2 5 9 )

s t a te ' X - r a y m a p p i n g o f s e le c te d e l e m e n t s

p r o v i d e d a u s e f ul a i d fo r c o m p a r t m e n t

i d e n t i f i c a t i o n i n f i ' o z e n - h y d r a t e d s e c t i o n s ' .

T h e y p r e s e n t p h o s l S h o r u s m a p s ( t h e ir F i g .

1 2) ' s u i ta b l e f o r p r e l i m i n a r y . . , i d e n ti f ic a -

t i o n ' o f n u c l e a r , c y t o p l a s m i c a n d e x t r a -

c e l l u l a r c o m p a r t m e n t s . H o ~ v e v e r , t h e y d o

n o t s t a t e t h e t i m e n e e d e d f o r e a c h m a p .

G i v e n t h e s m a l l d i ff e re n c e a n d t h e n o n -

u n i f o r m i t y o f p h o s p h o r u s c o n c e n t ra t i o n s i n

n u c l e u s a n d c y t o p l a s m , a n d v e r y l i m i t e d

X - r a y i n p u t r a t e t o l e ra t e d b y a E D S s p e c t r o -

m e t e r , t h e t i m e n e e d e d f o r s t a t i s t i c a l l y v a l i d

m a p s w i l l l e a v e l it t le t i m e f o r q u a n t i t a t i v e

a n a l y s is . S u c h p h o s p h o r u s m a p p i n g w o u l d

b e q u i t e i m p r a c t i c a l a s a m e a n s o f d i s t i n -

g u i s h i n g n u c l e u s a n d c y t o p l a s m i f m i c r o -

a n a ly s i s is i n t e n d e d f r o m t h e s a m e c r y o -

s e c t i o n s .

Reliability of restdts

( i) A s n o t e d a b o v e , t h e c a r d i n a l t e s t f o r r e li -

a b i l i t y o f a n y m i c r o a n a l y t i c a l p r o c e d u r e f o r

f r o z e n ' h y d r a t e d s e c t i o n s i s t h e a n a ly s i s O f

l a r g e e x t r a c e l l u l a r f r e e - f l u i d s p a c e s f r o m

t i ss u e s s u c h i n s e c t M a l p i g h i a n t u b u l e s

( G u p t a , ~ 19 76 ; G u p t a e taL , 1 9 7 6 , 1 9 7 7 ) o r

s a l i v a r y g l a n d s ( G u p t a et aL, 1 9 7 7 , 1 9 7 8 a ;

G U P T A A N D F 1 A LL

G u p t a a n d H a l l, 1 9 8 1 a ; F i g . 15 ) o r a n y o t h e r

f lu i d t r a n s p o r t i n g t i s s u e w h e r e t il e c o n t e n t s

o f t h e t r a n s p o r t e d f l u id c a n h e r e ad i l y

v e ri fi ed b y o t h e r s t a n d a r d m e t h o d s , i n

Callipho~t s a l i v a r y g l a n d s t h e m i c r o p r o b e

r e s u l t s h a v e b e e n c h e c k e d w i t h p a r a l l e l

n l e a s u r e m e n t s u s i n g K a n d C l s e le c t iv e

m i c r o e l e c t r o d e s b o t h f i n d e r n o r m a l c o n d i -

t i o n s ( G u p t a et aL, 1 9 7 8 a ; G u p t a & H a l l ,

1 97 9) a n d d u r i n g t r a n s p o r t i n a h y p o t o n i c

m e d i u m ( G u p t a a n d H a l l , 1 9 81 a , F i g . 1 5 a, b ).

O u r r e su l ts o n t h e M a l p i g h i a n t u b u l e s o f

Calliphora

( G u p l a , 1 9 76 ; G u p t a

et aL,

1977)

a n d Rhoththts ( G u p t a et oL, 1976, 19 ' /7 ) have

b e e n c o n f i r m e d b y M a r s h a l l ( 1 98 0 ) u s i n g

h y d r a t e d f l ' e e z e - f r a c t u r e d b u l k s a m p l e s . O u r

r e s u l t s o n

ht ~qtro

p r e p a r a t io n s o f t o a d

u r i n a r y b l a d d e r ( C i v a n

et al.,

1 9 8 0 ) a r e i n

v e ry g o o d a g r e e m e n t w i th t h e m i c r o p r o b e

r e s u l t s o f R i c k et aL ( 1 9 7 8 ) o n f r o z e n - d r i e d

s e c t i o n s . O u r o b s e r v a t i o n s o n t h e l o w l e v e l s

o f C a i n t h e m i t o c h o n d r i a o f n o r m a l c el ls

( G u p t a et aL, 1 9 7 8 a ; G u p t a a n d H a l l, 1 9 7 8)

a n d o n t h e l ow N a l ev e ls in n u c l e i ( G u p t a

et aL, 1 9 7 6 , 1 97 7 , 1 9 7 8 a , b ; J o n e s et aL,

1 9 7 9 ; F o r e r et aL, 1 9 8 0 ) a r e c o n s i s t e n t w i t h

o t h e r m i c r o p r o b e s t u d i e s es p e c ia l ly t h o s e b y

S o m l y o et aL ( 9 7 9 ) , b y R i c k et aL ( 1 9 78 ) a n d

b y B e c k et ol. ( 19 8 0 ). T h e c o n c l u s i o n t h a t t h e

n u c l e i h a v e i o n i c c o m p o s i t i o n s i m i l a r t o t il e

c y t o s o l , w i t h s l i g h t l y l e s s N a a n d s l i g h t l y

m o r e K h a s n o w b e e n c o n f i r m e d b y o t h e r

i n d e p e n d e n t t e c h n i q u e s ( s e e P a i n e et al.,

1 98 1) . T h e d a t a f o r t es s t h a n 3 0 ~ d r y m a s s

i n m o s t c e ll s a n a l y s e d b y u s , b y S o m l y o a n d

c o l l ea g u e s a n d b y R i c k a n d c o l l ea g u e s a r e

a l s o c o n s i s t e n t .

( ii ) T h e r e s u lt s f r o m t h e m i c r o p r o b e

s t u d i es b y S a u b e r m a n n a n d c o l l e ag u e s st il l

r e m a i n t o b e v e r if ie d . T h e i r e s t i m a t e s o f d r y

w t f r a c t i o n s i n th e r a t h e p a t o c y t e s ( S a u b e r -

m a n n a n d E c h l in , 19 7 5; S a u b e r m a n n et aL,

1 9 7 7 b ) a n d t h r e e c e ll t y p e s i n r e n a l p a p i l l a r y

t i p a r e i n t h e r a n g e o f 4 0 -5 0 .3 /o , s t r o n g l y

s u g g e s t i n g t h a t (a ) t h e i r ' f u l l y h y d r a t e d '

s e c t io n s m a y h a v e b e e n s u b s t a n t i a l l y d r y , a n d

( b) t h e i r m e t h o d s o f e s t i m a t i n g d r y m a s s

a r e n o t a c c u r a t e . I t is n o t e w o r t h y t h a t i f t h e

d r y w t f r a c t i o n s f o r t h e t h r e e c e l l t y p e s i n

t h e r a t r e n a l p a p i l l a ry t i p a r e c a l c u l a t e d f r o m

t h e d a t a i n S a u h e r m a n n et aL ( 1 9 8 1 a , - p .

2 6 7 , T a b l e I ) u s i n g ' M e t h o d B ' ( i. e. t h e r a t io s

o f R v a l u e s g i v e n i n t h e c o l u m n s ' H " a n d

" D ' a n d l e a v i n g o u t t h e e n t r i e s w i t h a l a r g e

p r o b a b l e e r ro r ) , o n e o b t a i n s t h e v a l u es

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M I C R O A N A L Y S I S O F F R O Z E N - H Y D R A T

Table 1 . D r y - w e i g h t f r a c t i o n s e s t h n a t e d f i ' o m

T a b le I o f S a u b e r m a n n et a l . ( 1 9 8 1 a ) . T h e e n t o '

f o r each e l emen t i s t he R-vah t e ' , pea k co t t n t/

con thuam t , f in t he hydra t ed s t a t e d i v i tl ed by t he

' R - v a h c e ' f b r t h e d e h y d r a t e d s t a t e ( ' A h , t h a d B ' ,

p . 636 )

Cel l type

C o l l e c t i n g P a p i l l a r y

duc t In te r s t i t ia l ep i the l ia l

N a ~ 0 . 1 3

P 0 . 3 1 0 . 3 5 0 . 3 2

S 0 . 4 3 ~ 0 . 4 2

C I 0 . 3 6 0 - 2 7 0 . 3 1

K 0 . 3 6 0 . 3 6 0 . 3 7

M e a n 0 . 3 7 0 . 2 8 0 . 3 6

s . d . 0 - 0 5 0 . 1 1 0 . 0 5

A v e r a g e o f a l l v a lu e s 0 - 3 3 - - + 0 . 0 8 .

l is te d h e re in T a b l e . T h e a v e r a g e o f n u m b e r s

i n t h i s t a b l e i s 3 3 . 2 % ( 2 8 % f o r i n t e r s t i t i a l

c e l ls ) . E v e n t h e s e a r e l i k e l y t o b e o v e r e s t i -

m a t e s i f t h e se c t i o n s h a d l o st s o m e w a t e r a t

t h e t i m e o f t h e m e a s u r e m e n t s a s s u g g e s te d

b y t h e i n a ag e d e t a i l s . I n v i e w o f t h e s e d a t a

i t i s d i ff i cu l t t o a c c e p t t h e c o r r e s p o n d i n g

v a l u e s o f 4 4 - 4 0 % a r r i v e d a t b y t h e a p p l i c a -

t i o n o f ' M e t h o d A ' i n t h e a r ti c le b y B u l g e r

e t a l . ( 1 9 8 1 ) . T h e s e d i s c r e p a n c i e s i n t h e e s t i -

m a t e s o f d r y w t f r a c t i o n s i n t r o d u c e a v e r y

l a r g e u n c e r t a i n t y i n t h e e s t i m a t e s o f e le -

m e n t a l c o n c e n t r a ti o n s i n m M p e r k g w e t w t

d e r i v e d i n d ir e ct l y f r o m t h e m e a s u r e m e n t s o n

f r o z e n - d r i e d s e c t i o n s .

T h e s e d o u b t s o n t h e a c c u r a c y o f th e r e su l ts

b y S a u b e r m a n n a n d c o l le a g u es h a v e b ee n

r e i n f o rc e d b y a r e c e n t m i c ' r o p r o b e s t u d y b y

B e c k

e t a L

( 1 9 8 1 ) o n t h e r a t r e n a l p a p i l l a

u n d e r a n t i d i u r e t i c c o n d i t i o n s ( u r i n e o s m o l a -

l it y 2 5 3 I + 3 4 2 m O s m / k g ) . T h e c o ll ec t in g

d u c t c e ll s h a d ( r a M k g - 1 w e t w t ) N a = 4 5 +

1 9; K = I 8 + 2 9 a n d C 1 = 8 7 _ 3 4 . T h e d ry

w t c o n c e n t r a t io n w a s 2 4 + _ 2 % o f w e t w t.

T h e s e v a l u e s a r e i n s h a r p c o n t r a s t w i t h t h e

c o r r e s p o n d i n g d a t a f r o m t h e c o ll e ct i ng d u c t

c e l l s b y B u l g e r

e t a L

( 1 9 8 1 ) ; N a = 3 4 4 + _ 1 2 7 ;

K = 1 5 6 _ . + 5 4 ; C 1 = 3 3 1 _ _ _ 1 0 8 ; d r y w t 4 3Y o

o f w e t w t . A b o u t 8 0 ~ h i g h e r v a l u e in t