The Scanning Electron Microscope: A High Information

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T H E ' 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 : A H I G H I N F O R M A T I O N C O N T E N T I M A G E O F B I O L O G I C A L ' S Y S T E M S

T. L. H A Y E S U N I V E R S I T Y O F C A L I F O R N I A , B E R K E L E Y

The p r inc ipa l value of t h e scanning e l ec t ron microscope (SEM) l i e s i n t h e instrument 's a b i l i t y t o produce high information content images. It i s not, i n general , an instrument of higher resolving power than t h e conven- t i o n a l transmission e l ec t ron microscope but t h e SEM can add usefu l informa- t i o n t h a t i s not ava i lab le i n t h e transmission e l ec t ron microscope image. The resolving power of current scanning e l ec t ron microscopes i s about 100 8.

The information r i c h i m a g e of t he SEM i s made possible by an imaging technique t h a t allows uncoupling of t h e two funct ions of a microscope: l o c a l i z a t i o n and information t r a n s f e r . The l i g h t microscope and the conven- t i o n a l transmission e l ec t ron microscope u t i l i z e a l e n s system f o r i m a g e formation. Such a system makes use of t h e same r a d i a t i o n t o both loca l i ze t h e poin ts of t h e specimen and t o ca r ry information about each point . A s a result, t h e parameters of r e so lu t ion and i m a g e information are l inked t o - gether by t h e type of r ad ia t ion used. For example, t he l i g h t microscope produces an image which i s r i c h i n information ( s p a t i a l , chemical, physio- l og ica l ) but low i n r e so lu t ion while t h e conventional transmission e l ec t ron microscope produces an image which i s high i n r e so lu t ion but low i n information (chemical bonds do not influence t h e image, t h e image i s t w o dimensional, t h e specimen i s not l i v i n g ) .

The S E M on t h e other hand uses a non-focused system of image forma- t i o n which allows t h e uncoupling of resolving power and information content. One kind of r ad ia t ion (e lec t rons) can be used t o l o c a l i z e t h e specimen poin ts at high r e so lu t ion while another kind of r ad ia t ion ( v i s i b l e l i g h t , secondaxy e lec t ron , e t c . ) can be used t o t r a n s f e r t h e information about these poin ts t o t h e image (Fig. 1). By u t i l i z i n g a t i m e sequence of po in ts (T.V.) as t h e imaging system, r a t h e r t han a l e n s focusing system, t h e SEM permits a g rea t increase i n t h e information transfer from specimen t o image.

This information r i c h image allows an inves t iga t ion of t h e second t r a n s f e r s tep: information t r a n s f e r from i m a g e t o observer. The usual mode of transfer i n s c i e n t i f i c s tud ie s m a k e s use of a reduct ion of t h e image t o ideas which are expressed i n words or numbers. i s t r ans fe r r ed by object ive, abs t r ac t means. The SEM can provide i m a g e s t h a t are su i tab le f o r t h i s type of t r a n s f e r as i l l u s t r a t e d by computer ana lys i s of images, p a t t e r n recogni t ion or chemical ana lys i s by cathodoluminescence or c h a r a c t e r i s t i c X-rays. The SEM can also provide images t h a t can help us t o inves t iga te another mode of information transfer from image t o observer: the subject ive, expe r i en t i a l mode. I n particularr, t h e secondary e l ec t ron mode of SEM operat ion produces an image with t h e important expe r i en t i a l f a c t o r of 3 dinensional space (Fig. 2) . world i s not su i ted t o o w expe r i en t i a l understanding but we sometimes fo rge t t h a t a 2 dimensional world i s equal ly fore ign . A t h ree dimensional image provides one important parameter t h a t permits t he image t o approximate experienced v i s ion (Fig. 3, 4) .

I n t h i s mode t h e information

W e are of ten aware tha t t h e four dimerisionjl

The value of expe r i en t i a l information t r a n s f e r depends somewhat on t h e d e f i n i t i o n of r e a l i t y t h a t we p re fe r . Many may not agree with Soren

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Kierkegaaxd when he suggests t h a t exis tence precedes essence but t he influence of 'his thoughts i s an e x i s t e n t i a l f a c t . It would seem t h a t at l e a s t we should inves t iga te t h e possible value of adding subject ive information t r a n s f e r t o the analyt ic , object ive modes which are d r e a d y familiar t o us. It i s possible t h a t i n addi t ion t o w r i t i n g t h e ana ly t ic program notes, we should t r y t o l i s t e n t o t h e symphony. i n biology (Fig. 5) subjective, expe r i en t i a l information t r a n s f e r w i l l add s ign i f i can t ly t o our understanding of t h e system.

P a r t i c u l a r l y i n deal ing with t h e very complex systems found

C onc l u s ion

We would suggest t h a t high information as wel l as high reso lu t ion should be considered i n t h e evaluat ion of a microscope's i m a g e and t h a t t he SEM i s a valuable instrument f o r t h e production of such high information con- t e n t images of b io log ica l systems. W e would a l so l i k e t o suggest t h a t sub- j e c t i v e as wel l as object ive cr i ter ia be considered when we evaluate the t r a n s f e r of t h i s information from image t o observer.

References

1) Hayes, T. L. and Pease, R. F. W. "The Scanning Electron Microscope: Pr inc ip les and Applications i n Biology and Medicine", i n Advances i n Biological and Medical Physics, (J. Lawrence and J. Gofman, eds.) Vol. 12, pp. 85-137, Academic Press, New York, 1968.

2) Microscopy", i n Advances i n Elec t ronics and Electron Physics (L. Marton, ed.) Vol. 21, pp. 181-247, Academic Press, New York, 1965.

Oatley, C. W., Nixon, W. C . and Pease, R. F. W. "Scanning Electron

3 ) Everhart , T . E., Wells, 0. C . and Oatley, C . W. "Factors Affecting Contrast and Resolution i n the Scanning Electron Microscope" , J. Elec t ronics and Control - 7,97-1ll, 1959.

4) Pease, R. F. W. "The Scanning Electron Microscope", lEEE Spectrum - 4, NO. 10, 96-102, O c t . 1967.

Ac knowle dgxe n t s

The Scanning Electron Microscope Program at Berkeley has been supported by t h e United S t a t e s Atomic Energy Commission, t h e J o i n t Services Elec t ronics Program, t h e United S ta t e s A i r Force Avionics Laboratory and the National I n s t i t u t e s of Health (Grant # GM5536-01).

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ELECTRON GUN

ELECTRON LENS

DEFLECTION

ELECTRON LENS

Secondary electrons

Characteristic x rays

Transmmed electrons or specimen current

SCANNING ELECTRON MICROSCOPE SCHEMATIC DIAGRAM

DEL 676-1648

Figure 1. Scanning e lec t ron microscope. Schematic diagram.

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Figure 2. Human red blood cells in blood clot. McDonald, L. W. and Hayes, T. L., Exp. and Mol. Path. - 10, 186-198, 1969. X 10,000.

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Figure 3. Living flour beet le . Pease, R . F. W., Hayes, T. L., Camp, A. S. and Amer, N. M., Science - 154, 1185, 1966. X 700.

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Figure 4. Eosinophil from peripheral blood of leukemic pa t i en t . McDonald, L. W. and Hayes, T. L., Exp. and Mol. Path. - 10, 186-198, 1969. x 2,000.

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Figure 5. Trabeculas meshwork of human eye, Spencer, W. H., Alvarrado, J. and Hayes, T. L., Invest igat ive Ophthallmology 7, 651-662, 1968. X 3,200.

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T. L. HAYES: Paper I n .

GUENTHER: Thank you very much D r . Hayes f o r showing once again t h a t progress i n t h e b io log ica l a rea i s so dependent upon advances i n physical sciences. To lead the discussion, Dr. Don DeFremery of t he Western U t i l i z a - t i o n Lab a t fibany. Don.

DON DeFREMERY: Thank you, M r . Chairman. I c e r t a i n l y want t o thank the three speakers f o r t h e s t imulat ing t a l k s t h a t they gave t o us. D r . Sayre f o r t he discussion of t he mechanical fragmentation of chicken myofibr i ls as influenced by th ree general types of treatments--heating or beating t o acce lera te glycolysis ; nembutal anesthesia or e l e c t r i c stunning t o retaxd post-mortum glycolysis ; and ioda-acetate or epanephrin treatment t o e l iminate glycolysis , and an attempt t o co r re l a t e t h e degree of fragmentation with the meat tenderness. Next we heard from D r . Thomas who gave us f i r s t a h i s to ry of microincineration of muscle as pr imit ive as it w a s i n the beginning and then a descr ip t ion of h i s own work on several facets--development of t he technique of low temperature ashing or low temperature inc inera t ion and i t s appl ica t ion t o b a c t e r i a l spores, p lan t viruses , mitochondrial p a r t i c l e s , var ious p l an t t i s s u e s even t o human h a i r . times, and f i n a l l y I should say we then heard f rom Dr. Hayes and from t h e t i t l e of h i s t a l k you might be l e d as t ray . a t ion t r a n s f e r and the perception of r e a l i t y by way of t h e scanning e l ec t ron microscope. The t i m e i s running a l i t t l e late, so I don ' t th ink I ' m going t o say very much more. I don ' t want t o pass up t h e opportunity, however, t o ask Dick Thomas one question -- a quest ion t h a t I thought needed answering. Dick, could you give us t h e manufacturers' names and the approximate p r i ces of several of t h e low temperature ashers t h a t you spoke of i n your t a l k ? F i r s t , s ince we both work f o r t h e U.S.D.A., perhaps I ought t o read our l e g a l disclaimer at t h i s po in t . o r recommendation of t h e product by t h e U. S. Department of Agriculture t o the exclusion of others t h a t may be su i tab le ."

I'd l i k e t o t a l k t o you Dick a l i t t l e b i t about human h a i r one of these

I would c a l l it a descr ip t ion of inform-

"Reference t o a company or product name does not imply approval

DICK THOMAS: Well ac tua l ly there are four companies t h a t I know of now. The f i r s t company t o ge t i n t o t h i s w a s Tracer Lab, t he d iv i s ion i n Richmond, Cal i forn ia , and they put out an instrument ca l led the low temperature asher, t h a t ' s been out f o r about six years now. The idea caught on and Coleman Instruments i s now of fer ing a device t h a t ' s ca l l ed a Model 40 R F Reactor, I bel ieve. A company, j u s t recent ly s taxted i n Hayward, ca l l ed In t e rna t iona l Plasma Corporation now o f f e r s an instrwnent. And then las t year I learned of a company i n Europe--Baltzer's Lippenstein t h a t also makes an instrument which w a s designed by a group i n Austr ia and in t e re s t ing ly enough, f o r me at least , i s intended pr imari ly f o r e l ec t ron microscope specimens. The p r i ces of these instruments, as competition has moved in, t h e p r i ces come down. They a l l run around $2,000 - $3000 or something l i k e t h a t .

DON DeFEIEMERY: Are the re any questions from the f l o o r ? I think, perhaps, t he speakers have overwhelmed you. I had a f e e l i n g a f t e r D r . Hayes's t a l k t h a t I w a s coming back t o t h e world or r e a l i t y as I knew it before, and I ' m not qu i te sure now, j u s t what r e a l i t y i s . I want t o thank p a r t i c u l a r l y t h e l as t two speakers f o r t h e qua l i t y of t h e e l ec t ron micrographs or t he scanning e l ec t ron micrographs, i f you w i l l . qua l i ty . D r . Guenther. John.

They were c e r t a i n l y of very exce l len t If t h e r e ' s no f u r t h e r discussion I'll t u r n the meeting back t o

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J. GUENTHER: Thanks Don. I guess we're about ready t o t u r n it back t o Max f o r t h e business session. L e t ' s stand up and take a break. Incidental ly , we did get a few l e t t e r s someone brought up here. There's one for Zerle Carpenter, f o r King or Landmann, Alsmeyer, Tuna, Kinsman and t h e r e ' s a postage due here for James Price. take a break.

Bet te r come back and ge t them. L e t ' s

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