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Introduction

The use of t h e scanning e lec t ron microscope (SEM) i n biology i s becoming widespread. It i s an excel lent t o o l f o r studying the 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 the op t i ca l and conventional e lec t ron microscope. I t ' s depth of f i e l d i s at least a hundred t i m e s g rea te r than t h a t of t he opt ica l microscope. objects may be studied with minimal preparation. For example, some material may be f ixed i n glutaraldehyde, dehydrated i n alcohol, a i r dr ied and then coated with a t h i n (200 Angstroms) f i lm of gold. This preparation i s much simplier than t h a t used i n conventional e lec t ron microscopy and avoids the necessi ty of t h i n sectioning the specimen.

Many

The use of t h e scanning e lec t ron 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. grea t deal, t h i s microscope has become a very useful instrument.

Since the b io logis t depends on morphology a

The Basic Instrument

A stream of e lec t rons i s scanned across the surface of the biological material i n a vacuum. This primary beam generates secondary electrons on the specimen surface which are gathered i n t o an e lec t ron col lector , point by point, as t h e material i s scanned. The magnitude of t h i s secondary co l lec t ion i s converted t o in t ens i ty of l i g h t as displayed on a cathode ray screen.

A more de ta i led account of the instrument i s a s follows. Electrons are emitted from an e lec t ron gun at the top of t h e instrument and are accelerated towards t h e anode which i s at ear th po ten t i a l r e l a t i v e t o the 2 t o 30 k i lovo l t s supplied t o t h e e lec t ron gun (see Figure 1). passes through one o r more magnetic l ens so t h a t t h e specimen i s bombarded w i t h a f i n e stream of e lectrons. generator which allows the beam t o successively scan the complete specimen surface. surface of t h e specimen which are then e l e c t r i c a l l y a t t rac ted t o a co l lec tor . The magnitude of t h e co l lec tor s ignal i s amplified and fed t o a cathode ray tube. The br ightness of i t s beam i s re la ted t o t h e number of secondary electrons col lected. scanning across t h e display tube such t h a t an image of t he specimen i s displayed. The inherent noise i n t h e system can be decreased by increasing the time of scanning. grea te r than t h a t used f o r t he monitor display tube.

The e lec t ron beam

The electron beam i s deflected by a scanning

The primary e lec t rons produce lower energy secondary electrons at t h e

The e lec t ron beam scanning of t he specimen i s coupled t o a s i m i l a r

Thus, f o r better pictures, t h e photographic exposure time i s much

Specimens several centimeters i n diameter may be placed i n t o the instrument. A manipulator allows the surface t o be t rans la ted and ro ta ted respective t o the impinging e lec t ron beam. most b io logica l specimens m u s t be dr ied before being used.

Since the specimen i s i n vacyum, A t h i n (200 A )

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coating of gold i s evaporated over t he surface of t he specimen t o increase secondary e lec t ron emission and a l s o t o leak off t he e l e c t r i c a l charge generated by the scanning e lec t ron beam.

Radiation other than secondary e lec t rons are a l so given off by the specimen. These can a l so be put t o g o d use. For example, the amount of cathodal luminescence released by the specimen i s r e l a t ed t o the amount of f luorescent mater ia ls within a given region of t he specimen. A par t of the e lec t ron beam t h a t penetrates the specimen may a lso be col lected benezth it so as t o simulate a conventional transmission microscope with poor resolution. However, t he re i s sone advantage i n obtaining simultaneously a transmitted image and a surface i m a g e from t h e same piece of biological mater ia l .

It i s of ten convenient t o obtain images from two d i f f e ren t angles t o

Stereoscopic viewers obtain a th ree dimensional picture of t he specimen. This i s very useful. when looking i n t o holes or crevices of biological s t ruc tures . have been u t i l i z e d t o make quant i ta t ive measurements with a good deal of accuracy. It should be noted, however, t h a t even without t he use of special stereoscopic techniques, all scanning e lec t ron microscope p ic tures have a three dimensional appearance due t o t he great depth of focus.

T i s sue Prepar a t ion

A s i n conventional histology, t he preparation techniques depend upon the pa r t i cu la r type of b io logica l material used. The surface of so f t t i s s u e i s usual ly washed with a j e t of isotonic solut ion although the removal of mucus may require special treatment. been developed i n our laboratory (Beidler, 1969). specimens are t r ea t ed as follows.

Many f i x a t i o n and drying methods have However, most s m a l l t i s sue

The t i s s u e (1 cm diameter or less) i s placed for 2 4 hours i n 6.25% gluteraldehyde made i n buffered sucrose. t i s s u e i n a series of ethanol-water solutions (50, 70, 80, 90, 95 and loo$) f o r i n t e rva l s of 2 hours each. The alcohol i s then replaced by amyl acetate by placing the t i s s u e i n 25, 50, 75, and 100% amyl acetate-ethanol solut ions 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 the amyl acetate i s replaced by l i qu id C02 and then heated t o above 3loC, the c r i t i c a l point of COz , so t h a t t he l i qu id CO2 becomes a gas and i s released from the t i s s u e without t i s s u e d i s to r t ion . Figure 2, 3) f o r C 0 2 c r i t i c a l point drying has been designed by M r . Webbers of our laboratory using t h e pr inc ip les outlined previously by Anderson (1951) and exploited for c e r t a i n scanning e lec t ron microscopic appl icat ions by Horridge and Tamm (1969).

Water i s replaced by placing the

A special apparatus (see

The t i s s u e i s placed i n a s m a l l s t a in l e s s s t e e l container (5) with a bottom of s t a in l e s s s t e e l wire c lo th . It i s then closed with a cover made of s t a in l e s s steel w i r e c loth. This con- t a i n e r i s placed i n a high pressure chamber made of s t a in l e s s steel about 2" on a side, sealed with an "Of' r ing (3) and the t o p ( 2 ) aff ixed with four socket screws. The pressure chamber i s connected on one side (6) with a CO2 cylinder v i a a valve. The other s ide (4) i s connected t o atmospheric pressure by means of a regulat ing valve and an on-off valve. gauge i s a l s o connected (8) t o the chamber. By opening i n l e t (6) and regulating the flow from ou t l e t (4), t he amyl acetate i n the t i s s u e i s replaced by l i qu id C 0 2 at room temperature.

A pressure

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When completely f i l l e d with l i qu id C02, t he ou t l e t (4 ) and i n l e t (6) are closed and t he temperature of t he chamber i s ra i sed by a b u i l t - i n car t r idge heater (1) t o a temperature of 5OoC. A t t h i s temperature t h e pressure i n the chamber i s 1400 PSI. The l i q u i d C02 is changed t o a gas. The ou t l e t (4) i s slowly and continuously opened over a period of 15 gas t o escape. The s low escape i s designed t o avoid a des t ruc t ive pressure gradient within the t i s s u e .

minutes f o r t he CO2

The dr ied t i s s u e i s placed with Duco cement onto a conventional aluminum stub and then coated with Palladium-gold (40:60) i n a Denton high vacuum evaporator. The stub i s then placed i n t o the scanning e lec t ron micro- scope 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 sh pars ley 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 he 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 these materials, but the above general procedure was followed. It i s hoped t h a t t he results shown w i l l i l l u s t r a t e some of t he 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 he study of foods using the scanning e lec t ron microscope.

A low magnification SEM photograph of the center of t h e raw potato i s shown i n Figure 4. A b e t t e r c e l l u l a r representat ion i s given i n Figure 5 where the individual s ta rch granules may now be seen with c l a r i t y . A photo- graph of s imilar type of t i s s u e and s imilar magnification (see Figure 6) was taken from a publ icat ion by Reeve (1967) where he used conventional h i s to- l og ica l procedures and polar iz ing opt ica l microscope. There i s a great d i f f e r - ence i n the type of information one may obtain with the S E M versus the opt ica l microscope. Note the three dimensional aspect of t h e SEM photograph. By merely turning a switch, a higher magnification of one group of s tarch 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 resolut ion a t above 30,000.

Untreated s t e w beef i s shown i n Figure 9. Note t h e la rge amount of connective t i s s u e and collagen. Individual bundles of f i b e r s are usual ly enclosed by a sheath as shown i n Figure 10 . Sometimes past of t h e sheath has been mechanically disturbed when we open the muscle although usual ly 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 the individual, ske l e t a l f ibers 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 he connective t i s s u e i s not apparent nor i s much of t he 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 he muscle f i b e r s (Figure 1 4 ) . are shown at magnifications of 5,000 i n Figure 15. It i s in te res t ing t o note t h a t when these f i b e r s are damaged they appear t o break at the 2-bands.

The A and I-bands

Parsley leaves have been selected f o r i l l u s t r a t i o n since the t i s s u e i s qui te d i f f e ren t from t h a t of beef o r potato. s m a l l por t ion of the leaf a t low magnification.

Figure 16 i l l u s t r a t e s a A t s l i g h t l y higher magnification

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t he stoma become apparent, as shown i n Figures 1 7 and 18. A t 8 magnifica- t i o n of 5,000, t he s t ruc tures of the stoma are 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 indicated some of t he p o s s i b i l i t i e s t h a t ex i s t i n the study of foods with the SEM. Ny own laboratory i s pr imari ly in te res ted i n t h e chemical receptors i n a va r i e ty of d i f f e ren t animals, including man. Special techniques may be developed f o r spec i f ic t i s s u e s . For example, one may need t o study the surface of t he human tongue over a period of several months. This can e a s i l y be accomplished by coating t h e surface of the tongue with s i l i cone rubber and making a mold of t he na tura l tongue surface. A posi t ive of the mold i s made, then coated with gold and examined with the scanning e lec t ron microscope. This can be done day after day without any discomfort t o the subject.

I cannot emphasize too strongly t h a t research f o r methods t o study b io logica l mater ia l i s s t i l l i n i t s infancy. The scanning e lec t ron microscope i s i n many ways easier t o use than the conventional e lec t ron microscope. It has been g rea t ly used i n the t e x t i l e and e lec t ronic indus t r ies i n a ra ther rout ine manner and there i s no reason why it cannot be adapted t o the food ind u s t r ie s .

Ac knowledaement s

The c l a r i t y of t he photographs i s due t o the superior prepnration of t he t i s s u e by Mrs. Ann Branden and the excel lent operation of t he SET.': by M r . Ron Parker.

This work i s supported i n par t by t h e National Science Foundation Research Grant GB-4068 and the National Science Foundation Science Development Grant t o F lor ida S ta t e University.

References

Anderson, T . F. (1951) Techniques f o r t he preservation of th ree dimensional s t ruc ture i n preparing specimens f o r t h e electronmicroscope. Trans. N. Y . Acad. Sci . Ser. 11, - 13, 130.

Beidler, L . M . (1969) The use of the SEM i n sensory biology. Second Annual E n g i s Symposium on SEM, 35.

Horridge, G. A. and T m , S. L. (1969) C r i t i c a l point drying f o r scanning e lec t ron 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 ta rch granules i n f r e sh potatoes. American Potato Journal, 44, 41. -

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Elec t ron G u n

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Amplif ier

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Display Cathode Ray

Tube

Specimen E lec t ron C o l l e c t o r

Figure 1. The basic instrument.

325

Figure 2. Photograph of C02 cr i t ical-point drying bomb together with valves controlling i n l e t s and out le ts .

326.

Figure 3.

I

Drawing of bomb showing mthcd of construction. d e t a i l s . See t ex t f o r

Figure 4. SEM photograph of s l i ce of raw potato. Magnification of 5ZX.

327.

Figure 5. Potato s l i ce magnified 210X. Notice starch granules i n each compartment.

Figure 6. Pmenchyma of water care of potato shown af'ter his tological preparation and viewed with polarizing microscope at 150X.

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Figure 7 . Enlargement of group of starch granules at 1050X.

Figure 8. A single starch granule at 4000X.

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Figure 9. Commercial stewing beef showing connective t i s sue and collagen 500X.

Figure 10. Single muscle bundle of beef showing ruptured sheath. 11OOX.

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Figure 11. A bundle of muscle f ibers mechanically damaged. 11OOX.

Figure 1 2 . Enlazgemnt of Figure 11 showing s t r ia ted muscle f ibers beneath sheath. 5500X.

331.

Figure 13. Cut end of stewing beef "Protem" treated. muscle. l O O X . Note how clean is the

Figure 14. Disrupted bundle of t reated beef muscle showing s t r i a t ions of f i b e r s . 21OOX.

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Figure 15. Muscle fiber striations showing prominent Z bands and fa in t A and Ibands. 50OOX.

333.

Figure 17. Underside of parsley leaf. 18OX.

Figure 18. Enlargemnt of leaf t o show numerous stoma. 500X.

334.

Figure 19. One of the parsley lea9 stoma magnified 500OX.

335.

J. D. SINK: We w i l l open t h e sess ion f o r d i scuss ion from the f l o o r again reminding you t o use one of t h e aisle mikes. S t a t e your n,me, i n s t i t u t i o n and then t h e question. Any questions?

Ti. K. JOHNSON: What i s t h e cos t of t h e ap;iTAxs?

L o M. BEIDLER: Now I ' m not here t o s e l l t h i s ins t ruxent yGi;.

under s t rind.

V. K. JOHNSON: Well I know t h a t , but I ' m i n t e r e s t e d .

L. M. BEIDUR: Well, when w e got our instrument it w a s around 60 thousand d o l l a r s , which i s t h e cos t of a very good transmission microscope. I th ink t h e Gerocol, ( ? ) i s also cheaper than the Cambridge.

J. D. SINK: Other comments. Questions?

UNIDENTIFIED: What kind of treatment d id you subject t he mater ia l t o vhen t h e collogen disappeared from t h e f i b e r s ?

L. 14. BEIDLER: Let 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 go t t en t h e same p ic tu re s . I was t a l k i n g t o t h i s group so 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 Pro ten- t rea ted . It w a s t h e Proten-beef, t h a t d id not have much connective t i s s u e around it. I used t h i s ma te 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 research on t h i s meat and I don ' t want t o ac t as if I ' m an exper t meats researcher or anything l i k e t h a t . off t h e she l f , t h i s meat.

So t h i s w a s r i g h t

UNIDENTIFIED: What i s t h e d i f fe rence between the e l e c t r o n micro- scope and t h e stereoscan e s p e c i a l l y i n t h e prepara t ions and i n t h e magnifi- c a t ion?

L. M. BEIDLER: Well, t h e usable magnification of t h e stereoecan i s about 30,000 with t h e transmission maybe a mi l l i on although people go up normally t o about 200,000, so it conventionally i s higher. The b ig d i f fe rence i s i n r e so lu t ion . I n ease of prepara t ion f o r t h i s instrument -- you can take a b u t t e r f l y and put it i n t h e instrument without any preparation, o r you can gold coat it. You can go from t h a t extreme t o very sophis t ica ted techniques. You could take fornaldahyde-treated s o f t t i s s u e , dehydrate it with alcohol, a i r dry it and put it i n t h e instrument, no microtoming, no sec t ion ing o r anything l i k e t h a t . about nodern techniques could use t h e instrument very very well . Now if you go t o t h e transmission e l e c t r o n microscope, as you probably know, it t a k e s a. long time t o l e a r n t o use t h e instrument. microtone proper ly and all t h e o ther techniques. So t h i s i s a simple i n s t r u - ment compared t o t h e transmission microscope.

A conventional morphologist who doesn ' t know anything

It t a k e s a long time t o l e a r n t o

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 th icknesses of t i s s u e s such as t h e sarcolemma or some other smaller item?

L. M. BEIDLER: I don ' t t h i n k it would be too good f o r thickness, un less you. could ge t it on an edge because t h e e l ec t rons generate t h e i r secondary e l e c t r o n s i n very t h i n shee ts on t h e surface. I n o ther words, it