Scanning transmission electron microscopy of biological macromolecules

I ]6

C.L. Woldringh, ed./Abstracts of electron microscopy conference

protein m o l e c u l e s placed on the apex of field emitter tips. They cover the metal tip as well as the protein molecules with a thin layer of benzene. Benzene desorbs quite easily from a protein b a s e ' a n d p r e l i m i n a r y results are repor t e d of imaging Bovine Serum Albumin (0.5 V/k, at 80 K). A n o t h e r FIM atom probe limitation is the small s a m p l i n g area. However, by s u c c e s s i v e evaporation it is p o s s i b l e to m a k e a 3-D elem e n t a l analysis as was r e p o r t e d by R. W a g n e r in a study on the m e c h a n i c a l h a r d e n i n g of Cu-Ti alloys. He was able to identify the p r e c i p i t a t i o n m e c h a n i s m as a spinodal one by m e a s u r i n g m o d u l a tion amplitudes and w a v e l e n g t h with the FIM atom probe. These points and some related papers p r e s e n t e d at the 9th Int. Congress on E lect r o n M i c r o s c o p y (Toronto, 1978) and the 25th Int. F i e l d E m i s s i o n S y m p o s i u m (Albuquerque, 1978) will be discussed.

ULTIMATE RESOLUTION MICROSCOPY

IN E L E C T R O N

A. M. J. Huiser Techn.

Phys.

Lab.,

Univ.

Groningen

In high r e s o l u t i o n m i c r o s c o p y the objects one deals w i t h are p h a s e objects. Since the i n t r o d u c t i o n of c o n t r a s t transfer theory in e l e c t r o n m i c r o s c o p y , it is known that phase c o n t r a s t only obtains by virtue of aberrations. The p a r a d o x i c a l C o n c l u s i o n is that high resolution electron microscopy requires aberrations. The c o n t r a s t t r a n s f e r f u n c t i o n depends, apart from the aberrations, also on the illumination. It is well known that the best t r a n s f e r is o b t a i n e d if the source is i n f i n i t e l y small (spatially c o h e r e n t illumination). A good a p p r o x i m a t i o n w o u l d be a field e m i s s i o n gun. The effect of the e x t e n t of the source is d i s a s t r o u s if the transfer f u n c t i o n rapidly oscillates, w h i c h will h a p p e n due to the e s s e n t i a l presence of a b e r r a t i o n s for high spatial frequencies. This e f f e c t i v e l y limits the a t t a i n a b l e resolution. It has always b e e n a s s u m e d that only the extent of the source affects the spatial r e s o l u tion. This p i c t u r e is rather naive since the s c a t t e r i n g process w i l l certainly also have influence on the spatial coherence. It has been shown b o t h exp e r i m e n t a l l y and t h e o r e t i c a l l y that especially inelastic scattering severely affeots the spatial coherence. The e f f e c t can be d e s c r i b e d by a b r o a d e n i n g of t h e source. However, also elastic s c a t t e r i n g i n f l u e n c e s the s p a t i a l co" h e r e n C e (caused by the fact that each

e l e c t r o n sees a d i f f e r e n t object, since the p a r t i c l e s in the o b j e c t move). The first p r o b l e m can be coped with by e n e r g y - s e l e c t i v e detection, e.g., in STEM. The second problem, however, cannot be c i r c u m v e n t e d and t h e r e f o r e u l t i m a t e l y limits the r e s o l u t i o n w h i c h can be achieved.

SCANNING TRANSMISSION ELECTRON MICROSCOPY OF B I O L O G I C A L M A C R O M O L E C U L E S W. Tichelaar, M. G. van Heel, E. F. J. van Bruggen

J. Haker,

Biochemisch Laboratorium der R.U.G., N i j e n b o r g h 16, 9747 AG G r o n i n g e n

We w a n t to study the p o s s i b l e advantages of the S c a n n i n g T r a n s m i s s i o n Electron M i c r o s c o p e (STEM) over the Conventional 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 (CTEM) w h e n a p p l i e d to b i o l o g i c a l macromolecules. Our Philips EM 400 is e q u i p p e d w i t h a F i e l d E m i s s i o n Gun and a S T E M - a t t a c h m e n t ; the i n s t r u m e n t is u s u a l l y o p e r a t e d in the dark field (df) mode. I P h o t o g r a p h s have been taken d i r e c t l y from the m o n i t o r screen, sometimes with i n v e r s e d contrast. The m o l e c u l e s were p r e p a r e d on a thin c a r b o n film and light n e g a t i v e l y stained. The f o l l o w i n g results are obtained: I. Resolution: a p o i n t - t o - p o i n t resolution of about 5 A was r e g i s t e r e d . 2. Small proteins: as expected, p a p a i n and IgG show a b e t t e r c o n t r a s t in STEM-df than in CTEM-bf. 3. P r o t e i n - n u c l e i c acid complexes: (a) S t r u c t u r a l d e t a i l of r o o s t e r liver p o l y r i b o s o m e s can be seen m o r e e a s i l y in STEM-df than in CTEM-bf. (b) The q u a l i t y of the STEM-df images of c o m p l e x e s of T 7 D N A - R N A p o l y m e r a s e is at least as good as in the case of CTEM-df. 4. P e r i o d i c a l objects: a t w o - d i m e n s i o n al c o m p u t e r r e c o n s t r u c t i o n was carried out of a STEM-df image of a f l a t t e n e d tubular P o l y m e r of H e l i x p o m a t i a 8 - h e m o c y a n i n 2 . In the rec o n s t r u c t e d image a t e n d e n c y for s p l i t t i n g up of the m o r p h o l o g i c a l units in domains can be seen. Resuming, we can c o n c l u d e that the app l i c a t i o n of the STEM to w e a k l y scattering b i o l o g i c a l objects looks promising.In the near future the STEM w i l l be c o u p l e d o n - l i n e to a m i n i c o m p u t e r system. Of this d e v e l o p m e n t we e x p e c t a s i g n i f i c a n t e n h a n c e m e n t of the e f f i c i e n c y of r e g i s t r a t i o n and an i m p o r t a n t i n c r e a s e in the n u m b e r of r e g i s t e r e d image points. i.

w. Tichelaar,

et al.

(1978),

Proc.