320.
Introduction
The use of t h e scanning e l e c t r o n microscope (SEM) i n biology i s
becoming widespread. It i s an e x c e l l e n t t o o l f o r studying t h e morphology of
c e l l s and t i s s u e s . I t ' s magnification, 20 t o 30 thousand times, overlaps both
t h e o p t i c a l and conventional e l e c t r o n microscope. I t ' s depth of f i e l d i s a t
l e a s t a hundred t i m e s g r e a t e r t h a n t h a t of t h e o p t i c a l microscope. Many
o b j e c t s may be studied with minimal preparation. For example, some material
may be f i x e d i n glutaraldehyde, dehydrated i n alcohol, a i r d r i e d and t h e n
coated with a t h i n (200 Angstroms) f i l m of gold. This preparation i s much
simplier t h a n t h a t used i n conventional e l e c t r o n microscopy and avoids t h e
n e c e s s i t y of t h i n sectioning t h e specimen.
The use of t h e scanning e l e c t r o n microscope i n biology i s s t i l l i n
i t s infancy. Sophisticated techniques must s t i l l be developed so t h e use of
t h e microscope w i l l be enhanced. Since t h e b i o l o g i s t depends on morphology a
g r e a t d e a l , t h i s microscope has become a very u s e f u l instrument.
The Basic Instrument
A stream of e l e c t r o n s i s scanned across t h e surface of t h e b i o l o g i c a l
material i n a vacuum. This primary beam generates secondary e l e c t r o n s on t h e
specimen surface which are gathered i n t o an e l e c t r o n c o l l e c t o r , point by point,
as t h e material i s scanned. The magnitude of t h i s secondary c o l l e c t i o n i s
converted t o i n t e n s i t y of l i g h t as displayed on a cathode ray screen.
A more d e t a i l e d account of t h e instrument i s a s follows. Electrons
a r e emitted from an e l e c t r o n gun a t t h e t o p of t h e instrument and are
accelerated towards t h e anode which i s a t e a r t h p o t e n t i a l r e l a t i v e t o t h e 2 t o
30 k i l o v o l t s supplied t o t h e e l e c t r o n gun ( s e e Figure 1). The e l e c t r o n beam
passes through one o r more magnetic l e n s s o t h a t t h e specimen i s bombarded w i t h
a f i n e stream of e l e c t r o n s . The e l e c t r o n beam i s deflected by a scanning
generator which allows t h e beam t o successively scan t h e complete specimen
surface. The primary e l e c t r o n s produce lower energy secondary e l e c t r o n s a t t h e
surface of t h e specimen which are then e l e c t r i c a l l y a t t r a c t e d t o a c o l l e c t o r .
The magnitude of t h e c o l l e c t o r s i g n a l i s amplified and f e d t o a cathode r a y
tube. The b r i g h t n e s s of i t s beam i s r e l a t e d t o t h e number of secondary e l e c t r o n s
c o l l e c t e d . The e l e c t r o n beam scanning of t h e specimen i s coupled t o a s i m i l a r
scanning across t h e d i s p l a y tube such t h a t an image of t h e specimen i s displayed.
The inherent noise i n t h e system can be decreased by increasing t h e time of
scanning. Thus, f o r better pictures, t h e photographic exposure time i s much
g r e a t e r than t h a t used f o r t h e monitor d i s p l a y tube.
Specimens s e v e r a l centimeters i n diameter may be placed i n t o t h e
instrument. A manipulator allows t h e surface t o be t r a n s l a t e d and r o t a t e d
respective t o t h e impinging e l e c t r o n beam. Since t h e specimen i s i n vacyum,
most b i o l o g i c a l specimens m u s t be d r i e d before being used. A t h i n ( 2 0 0 A )
321.
c o a t i n g of gold i s evaporated over t h e s u r f a c e of t h e specimen t o increase
secondary e l e c t r o n emission and a l s o t o l e a k o f f t h e e l e c t r i c a l charge
generated by t h e scanning e l e c t r o n beam.
Radiation o t h e r t h a n secondary e l e c t r o n s a r e a l s o given off by t h e
specimen. These can a l s o be put t o g o d u s e . For example, t h e amount of
cathodal luminescence r e l e a s e d by t h e specimen i s r e l a t e d t o t h e amount of
f l u o r e s c e n t m a t e r i a l s w i t h i n a given region of t h e specimen. A p a r t of t h e
e l e c t r o n beam t h a t p e n e t r a t e s t h e specimen may a l s o be c o l l e c t e d benezth it s o
as t o simulate a conventional transmission microscope with poor r e s o l u t i o n .
However, t h e r e i s sone advantage i n obtaining simultaneously a t r a n s m i t t e d
image and a surface i m a g e from t h e same piece of b i o l o g i c a l m a t e r i a l .
It i s o f t e n convenient t o o b t a i n images from two d i f f e r e n t angles t o
o b t a i n a t h r e e dimensional p i c t u r e of t h e specimen. This i s very useful. when
looking i n t o h o l e s or c r e v i c e s of b i o l o g i c a l s t r u c t u r e s . Stereoscopic viewers
have been u t i l i z e d t o make q u a n t i t a t i v e measurements with a good d e a l of
accuracy. It should be noted, however, t h a t even without t h e use of s p e c i a l
stereoscopic techniques, all scanning e l e c t r o n microscope p i c t u r e s have a t h r e e
dimensional appearance due t o t h e g r e a t depth of f o c u s .
T i s sue Prepar a t i o n
A s i n conventional histology, t h e p r e p a r a t i o n techniques depend upon
t h e p a r t i c u l a r type of b i o l o g i c a l material used. The surface of s o f t t i s s u e
i s u s u a l l y washed with a j e t of i s o t o n i c s o l u t i o n although t h e removal of
mucus may r e q u i r e s p e c i a l treatment. Many f i x a t i o n and drying methods have
been developed i n our l a b o r a t o r y (Beidler, 1969). However, most s m a l l t i s s u e
specimens a r e t r e a t e d as follows.
The t i s s u e (1cm diameter or l e s s ) i s placed for 2 4 hours i n 6.25%
gluteraldehyde made i n buffered sucrose. Water i s replaced by placing t h e
t i s s u e i n a series of ethanol-water s o l u t i o n s (50, 70, 80, 90, 95 and loo$) f o r
i n t e r v a l s of 2 hours each. The alcohol i s t h e n replaced by amyl a c e t a t e by
p l a c i n g t h e t i s s u e i n 25, 50, 75, and 100% amyl a c e t a t e - e t h a n o l s o l u t i o n s
f o r 1 0 minutes each. To avoid a r t i f a c t s caused by phase boundaries or c r y s t a l
formations t h e amyl a c e t a t e i s replaced by l i q u i d C02 and t h e n heated t o above
3loC, t h e c r i t i c a l p o i n t of COz, so t h a t t h e l i q u i d CO2 becomes a gas and i s
r e l e a s e d from t h e t i s s u e without t i s s u e d i s t o r t i o n . A s p e c i a l apparatus ( s e e
Figure 2, 3) f o r C 0 2 c r i t i c a l p o i n t drying has been designed by M r . Webbers of
our l a b o r a t o r y using t h e p r i n c i p l e s o u t l i n e d previously by Anderson (1951) and
e x p l o i t e d for c e r t a i n scanning e l e c t r o n microscopic a p p l i c a t i o n s by Horridge
and Tamm (1969).
The t i s s u e i s placed i n a s m a l l s t a i n l e s s s t e e l container (5)
with a bottom of s t a i n l e s s s t e e l wire c l o t h . It i s t h e n closed
with a cover made of s t a i n l e s s steel w i r e c l o t h . This cont a i n e r i s placed i n a high pressure chamber made of s t a i n l e s s
s t e e l about 2" on a side, sealed with an "Of' r i n g (3) and t h e
t o p ( 2 ) a f f i x e d with f o u r socket screws. The pressure chamber
i s connected on one side ( 6 ) with a CO2 c y l i n d e r v i a a valve.
The o t h e r s i d e ( 4 ) i s connected t o atmospheric pressure by
means of a r e g u l a t i n g valve and an on-off valve. A pressure
gauge i s a l s o connected (8) t o t h e chamber. By opening i n l e t
( 6 ) and r e g u l a t i n g t h e flow from o u t l e t (4), t h e amyl a c e t a t e
i n t h e t i s s u e i s replaced by l i q u i d C 0 2 a t room temperature.
322.
When completely f i l l e d with l i q u i d C02, t h e o u t l e t ( 4 ) and i n l e t
(6) are closed and t h e temperature of t h e chamber i s r a i s e d by
a b u i l t - i n c a r t r i d g e h e a t e r (1)t o a temperature of 5OoC. A t
t h i s temperature t h e pressure i n t h e chamber i s 1400 PSI. The
l i q u i d C02 i s changed t o a gas. The o u t l e t (4) i s slowly and
continuously opened over a period of 15 minutes f o r t h e CO2
gas t o escape. The s l o w escape i s designed t o avoid a
d e s t r u c t i v e pressure gradient within t h e t i s s u e .
The d r i e d t i s s u e i s placed with Duco cement onto a conventional
aluminum stub and t h e n coated with Palladium-gold (40:60) i n a Denton high
vacuum evaporator. The stub i s t h e n placed i n t o t h e scanning e l e c t r o n microscope and positioned properly f o r scanning.
Sample Photographs
Raw potatoes (Red B l i s s ) , untreated s t e w beef, "Protern"-treated stew
beef and f r e s h p a r s l e y leaves were selected f o r t h e i r possible i n t e r e s t t o
m e m b e r s of t h e American Meat I n s t i t u t e Conference. Special methods of t i s s u e
preparation were not researched f o r t h e s e materials, but t h e above general
procedure was followed. It i s hoped t h a t t h e results shown w i l l i l l u s t r a t e
some of t h e p o s s i b i l i t i e s t h a t e x i s t i n t h e study of foods using t h e scanning
e l e c t r o n microscope.
A low magnification SEM photograph of t h e c e n t e r of t h e raw p o t a t o
i s shown i n Figure 4. A b e t t e r c e l l u l a r r e p r e s e n t a t i o n i s given i n Figure 5
where t h e i n d i v i d u a l s t a r c h granules may now be seen with c l a r i t y . A photograph of s i m i l a r type of t i s s u e and s i m i l a r magnification ( s e e Figure 6) was
taken from a p u b l i c a t i o n by Reeve (1967) where he used conventional h i s t o l o g i c a l procedures and p o l a r i z i n g o p t i c a l microscope. There i s a g r e a t d i f f e r ence i n t h e type of information one may obtain with t h e S E M versus t h e o p t i c a l
microscope. Note t h e three dimensional aspect of t h e SEM photograph. By
merely t u r n i n g a switch, a higher magnification of one group of s t a r c h granules
i s obtained as shown i n Figure 7. A single granule i s shown i n Figure 8. This
i s a magnification of only 4,000 whereas t h e SEM can s t i l l give good r e s o l u t i o n
a t above 30,000.
Untreated s t e w beef i s shown i n Figure 9 . Note t h e l a r g e amount of
connective t i s s u e and collagen. Individual bundles of f i b e r s are u s u a l l y
enclosed by a sheath as shown i n Figure 1 0 . Sometimes past of t h e sheath has
been mechanically disturbed when we open t h e muscle although u s u a l l y t h i s sheath
i s i n t a c t . Figure 11 and 1 2 i l l u s t r a t e where a s m a l l portion of such a bundle
has been damaged and t h e individual, s k e l e t a l f i b e r s beneath are exposed.
"Protem"-treated s t e w beef i s much cleaner i n appearance as shown i n
Figure 13. Much of t h e connective t i s s u e i s not apparent nor i s much of t h e
collagen. If a single fiber bundle i s magnified t o over 2,000 t i m e s , one can
e a s i l y see t h e Z-bands of t h e muscle f i b e r s (Figure 1 4 ) . The A and I-bands
are shown a t magnifications of 5,000 i n Figure 15. It i s i n t e r e s t i n g t o note
t h a t when t h e s e f i b e r s are damaged they appear t o break a t t h e 2-bands.
P a r s l e y leaves have been selected f o r i l l u s t r a t i o n since t h e t i s s u e
i s q u i t e d i f f e r e n t from t h a t of beef o r potato. Figure 1 6 i l l u s t r a t e s a
s m a l l p o r t i o n of t h e leaf a t low magnification. A t s l i g h t l y higher magnification
523.
t h e stoma become apparent, as shown i n F i g u r e s 1 7 and 18. A t 8 magnificat i o n of 5,000, t h e s t r u c t u r e s of t h e stoma a r e e a s i l y observed (Figurc. 1 3 ) .
I hope t h e above i l l u s t r a t i o n s have i n d i c a t e d some of t h e
p o s s i b i l i t i e s t h a t e x i s t i n t h e study of foods with t h e SEM. Ny own
l a b o r a t o r y i s p r i m a r i l y i n t e r e s t e d i n t h e chemical r e c e p t o r s i n a v a r i e t y of
d i f f e r e n t animals, including man. S p e c i a l techniques may be developed f o r
s p e c i f i c t i s s u e s . For example, one may need t o study t h e surface of t h e
human tongue over a period of s e v e r a l months. T h i s can e a s i l y be accomplished
by c o a t i n g t h e surface of t h e tongue with s i l i c o n e rubber and making a mold of
t h e n a t u r a l tongue s u r f a c e . A p o s i t i v e of t h e mold i s made, t h e n coated with
gold and examined with t h e scanning e l e c t r o n microscope. This can be done day
a f t e r day without any discomfort t o t h e s u b j e c t .
I cannot emphasize t o o s t r o n g l y t h a t research f o r methods t o study
b i o l o g i c a l m a t e r i a l i s s t i l l i n i t s infancy. The scanning e l e c t r o n microscope
i s i n many ways easier t o use t h a n t h e conventional e l e c t r o n microscope. It
has been g r e a t l y used i n t h e t e x t i l e and e l e c t r o n i c i n d u s t r i e s i n a r a t h e r
r o u t i n e manner and t h e r e i s no reason why it cannot be adapted t o t h e food
ind u s t r i e s
.
Ac knowledaement s
The c l a r i t y of t h e photographs i s due t o t h e s u p e r i o r prepnration
of t h e t i s s u e by Mrs. Ann Branden and t h e e x c e l l e n t operation of t h e SET.':
by M r . Ron Parker.
T h i s work i s supported i n p a r t by t h e National Science Foundation
Research Grant GB-4068 and t h e National Science Foundation Science Development
Grant t o F l o r i d a S t a t e University.
References
(1951) Techniques f o r t h e p r e s e r v a t i o n of t h r e e dimensional
Anderson, T . F.
s t r u c t u r e i n preparing specimens f o r t h e electronmicroscope. Trans. N. Y.
Acad. S c i . Ser. 11, 13, 130.
Beidler, L . M .
(1969) The use of t h e SEM i n sensory biology.
E n g i s Symposium on SEM, 35.
Second Annual
Horridge, G. A. and T m , S. L. (1969) C r i t i c a l p o i n t drying f o r scanning
e l e c t r o n microscopic study of c i l i a r y motion. Science, 163, 817.
-
Reeve, R . M.
(1967) Suggested improvements f o r microscopic measurement of
c e l l s and s t a r c h granules i n f r e s h p o t a t o e s . American Potato Journal,
44, 41.
-
324.
Electron G u n
l!!J
I
Display
Cathode Ray
Tube
I
r
1
1Anode
I
-
>
Scanning
Gene r a t or
w
Amplifier
h
Specimen
Figure 1.
Electron
Collector
The basic instrument.
rl
325
Figure 2.
Photograph of C 0 2 c r i t i c a l - p o i n t drying bomb together with
valves controlling i n l e t s and o u t l e t s .
326.
I
Figure 3 .
Figure 4.
Drawing of bomb showing m t h c d of construction.
details
.
SEM photograph of s l i c e of raw potato.
See t e x t f o r
Magnification of 5ZX.
327.
Figure 5.
Potato s l i c e magnified 210X.
compartment.
Notice s t a r c h granules i n each
Figure 6.
Pmenchyma of water care of potato shown af'ter h i s t o l o g i c a l
preparation and viewed with polarizing microscope at 150X.
328.
Figure 7 .
Enlargement of group of s t a r c h granules a t 1050X.
Figure 8.
A s i n g l e s t a r c h granule at 4000X.
329.
Figure 9.
Figure 10.
Commercial stewing beef showing connective t i s s u e and collagen
500X.
Single muscle bundle of beef showing ruptured sheath. 11OOX.
330.
Figure 11.
Figure 1 2 .
A bundle of muscle f i b e r s mechanically damaged.
11OOX.
Enlazgemnt of Figure 11 showing s t r i a t e d muscle f i b e r s beneath
sheath. 5500X.
331.
Figure 13.
Figure 14.
.
Cut end of stewing beef "Protem" t r e a t e d .
muscle. l O O X
Note how clean is the
Disrupted bundle of t r e a t e d beef muscle showing s t r i a t i o n s of
fibers
21OOX.
.
332.
Figure 15.
Muscle fiber striations showing prominent Z bands and f a i n t A and
Ibands.
50OOX.
333.
Figure 17.
Figure 18.
Underside of parsley leaf.
18OX.
Enlargemnt of leaf t o show numerous stoma.
500X.
334.
Figure 19.
One of the parsley lea9 stoma magnified 500OX.
335.
J. D. SINK: W
e w i l l open t h e s e s s i o n f o r d i s c u s s i o n from t h e
f l o o r again reminding you t o use one of t h e a i s l e mikes. S t a t e your n,me,
i n s t i t u t i o n and t h e n t h e q u e s t i o n . Any q u e s t i o n s ?
Ti. K. JOHNSON:
L o M. BEIDLER:
under s t rind.
V. K. JOHNSON:
What i s t h e c o s t of t h e ap;iTAxs?
Now I ' m not here t o s e l l t h i s i n s t r u x e n t
yGi;.
Well I know t h a t , b u t I ' m i n t e r e s t e d .
L. M. BEIDUR: Well, when w e g o t our instrument it w a s around 60
thousand d o l l a r s , which i s t h e c o s t of a v e r y good t r a n s m i s s i o n microscope.
I t h i n k t h e Gerocol, ( ? ) i s also cheaper t h a n t h e Cambridge.
J. D.
SINK:
Other comments.
Questions?
UNIDENTIFIED: What kind of t r e a t m e n t d i d you s u b j e c t t h e m a t e r i a l
t o vhen t h e c o l l o g e n disappeared from t h e f i b e r s ?
L. 14. BEIDLER: L e t me say I d i d n ' t t a ; f f r e s h muscle. If I had I
doubt t h a t I'd have g o t t e n t h e same p i c t u r e s . I was t a l k i n g t o t h i s group s o
I went out t o a s t o r e and I bought stewing beef and I took stewing beef not
t r e a t e d and P r o t e n - t r e a t e d . It w a s t h e Proten-beef, t h a t d i d not have much
connective t i s s u e around it. I used t h i s m a t e r i a l as an i l l u s t r a t i o n f o r
t h i s group. I d i d n ' t do any r e s e a r c h on t h i s meat and I d o n ' t want t o a c t as
if I ' m an e x p e r t meats r e s e a r c h e r o r anything l i k e t h a t . So t h i s w a s r i g h t
o f f t h e s h e l f , t h i s meat.
UNIDENTIFIED: What i s t h e d i f f e r e n c e between t h e e l e c t r o n microscope and t h e s t e r e o s c a n e s p e c i a l l y i n t h e p r e p a r a t i o n s and i n t h e magnific a t ion?
L. M. BEIDLER: Well, t h e usable m a g n i f i c a t i o n of t h e stereoecan i s
about 30,000 with t h e t r a n s m i s s i o n maybe a m i l l i o n although people go up
normally t o about 200,000, so it c o n v e n t i o n a l l y i s higher. The b i g d i f f e r e n c e
i s i n r e s o l u t i o n . I n ease of p r e p a r a t i o n f o r t h i s instrument -- you can t a k e
a b u t t e r f l y and put it i n t h e instrument without any p r e p a r a t i o n , o r you can
gold c o a t it. You can go from t h a t extreme t o v e r y s o p h i s t i c a t e d techniques.
You could t a k e f o r n a l d a h y d e - t r e a t e d s o f t t i s s u e , dehydrate it with alcohol,
a i r d r y it and put it i n t h e instrument, no microtoming, no s e c t i o n i n g o r
anything l i k e t h a t . A conventional morphologist who d o e s n ' t know anything
about nodern t e c h n i q u e s could use t h e instrument v e r y v e r y w e l l . Now i f you
go t o t h e t r a n s m i s s i o n e l e c t r o n microscope, as you probably know, it t a k e s a.
l o n g time t o l e a r n t o use t h e instrument. It t a k e s a l o n g time t o l e a r n t o
microtone p r o p e r l y and all t h e o t h e r techniques.
So t h i s i s a simple i n s t r u ment compared t o t h e t r a n s m i s s i o n microscope.
R, L. HENRICKSON ( O k l a h o m a ) : How e f f e c t i v e i s t h e instrument f o r
measuring t h i c k n e s s e s of t i s s u e s such as t h e sarcolemma o r some o t h e r smaller
item?
L. M. BEIDLER: I d o n ' t t h i n k it would be t o o good f o r t h i c k n e s s ,
u n l e s s you. could g e t it on an edge because t h e e l e c t r o n s generate t h e i r
secondary e l e c t r o n s i n very t h i n s h e e t s on t h e s u r f a c e . I n o t h e r words, it