Fast-Scan EM With Digital Image ProcessingFor Dynamic Experiments

Charles W. McMillin

Fred C. BillingsleyRobert E. Frazer

ABSTRACT. The recent introduction of accessory instrumentation capable of display attelevision scan rates suggests a broadened application for the scanning electronmicro scope- the direct observation of motion Idynamic events I at magnifications otherwiseunattainable. In one illustrative experiment, the transverse surface of southern pine wasobserved when subjected to large compressive forces perpendicular to the grain. Tracheidwalls were seen to bend or distort skleways and ultimately rupture. In a second example, asingle tracheid of southern pine was shown to fail while under torsional stress and unwindinto a ribbonlike element-a structure that provides the coherence necessary for strengthdevelopment in refiner groundwood pulp. Complementing the dynamic applications of thefast-scan EM are newly developed techniques by which pictures can be stored, proceaaed,and displayed by a digital computer system. Digital data processing can sharpen picturecontrast. detect and display differences between pictures, and perform computationalanalysis.

indicate a few possibilities for digital imageprocessing.

Dynamic ExperimentsFigure 1 is a block diagram of system

modifications that permit visual observation.video recording. and photography of dynamicevents. The standard specimen stage (A) wasmodified with a variety of manipulativesubstages; a few examples are given later inthe text. Output of the secondary electrondetector photomultiplier (B) was fed through apreamplifier (C) ro a video distribution am.plifier (D). A scan generaror (E) deflected theelectron beam in synchronization with the

C. W. McMillin is on the staff of the SouthernForest Expt. Sta. USDA Forest Service, Pineville,La. F. C. Billingsiey and R. E. Frazer are with theJet Propulsion r;ab., California Inst. of Technolov,Pasadena, Calif. Aspects of digital image processmg

.di8a1ssed in this paper are the result of one phase ofresearch carried out at the Jet Propulsion Lab.under Contract NAS 7.100, sponsored by theNational Aeronautics and Space Administration.The authors acknowledge the assistance of D.A.O'Handley and A. R. Gillespie for the film scanningand F. G. Staudhammer for the computerprocessing. This paper was presented at a sym.posium on the Ultrastructure of Wood and WoodProducts, sponsored by the Biology and Pulp atPaper Technical Committees of the Forest ProductsResearch Society in cooperation with the USDAForest Service North Central and Southern ForeatExperiment Stations, February 26.28, 1973, inAlexandria, La. It was received for publication inAugust 1973.

One of the outstanding characteristics ofthe SEM image is its three-dimensionalquality. Since the introduction of commercialinstruments in 1965. microscopists haveeagerly depicted the structure of wood with aclarity usually lacking in micrographs made byother means.

Observations at slow scan rates are largelylimited to stationary surfaces. but recentlyintroduced instrumentation provides thecapability of display at television scan rates.This innovation allows the direct observationof motion (dynamic events) at magnificationsotherwise unattainable.

Techniques have recently been developed bywhich television pictures can be stored.processed. and displayed by a digi-tal computer system. Although the tech-nology has principally been applied toaerospace video-data. it may also prove ap-plicable in fast-scan EM. For example. TV -rateSEM images have relatively poor resolution.and computer processing can sharpen thepicture and emphasize details. In addition,digital SEM data may be manipulated toreveal differences between pictures and toperform computational analysis.

The purpose of this paper was to illustratetypical dynamic experiments that can be ac-complished with fast-scan EM, to discuss somedetails of the system design. and finally to

JANUARY 974272

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sideways and ultimately rupture. Figure 2shows a substage for observation of suchinelastic failure. The stage consists of a fixedhead (AI attached to a base plate (BI and amovable head (CI actuated by a pair ofprecision ball screws (D). The screws are drivenby a worm.gear reduction unit (E) connectedby shaft (F) to a slow.speed DC motor outsidethe specimen chamber. Oear ratios were suchthat one revolution of the external shaft ad.vanced the movable head 0.0033 inch. With theunit in place, the normal X, Y ,Z, and tiltmotions of the stage could be operated in theusual manner. The transverae surface of a 6.mm cube of 8Outhson pine (0) was exposed byhand sectioning and positioned between theloading heads 80 that compression was appliedto the radial walls of cells.

Figure 3 is a sequence of four micrographsof inelastic compressM>n failure. The com.pressive force is in a direction from top tobottom of each micrograph. In the undeformedtransverse surface (AI, cell walls and a singleray are clearly visible. Details on the cell wallsurface within the lumen are difficult todistinguish. The lower row of cells was used asa reference plane in each micrograph. Theupper surface of the specimen (that adjacent tothe movable loading head) was eight cell rowsabove the top shown in A. Micrograph B showsthe sample after a moderate degree of com.pression; the ray cell has collapsed, and in.dividual tracheids have distorted. The tracheidin the upper center has buckled and partiallycollapsed into the adjacent cell. Micrographs Cand D show additional distortion of tracheidsand progressive buckling and collapse asoompression increasee. Many fractures can beseen across cell walls as well as separationsbetween cells in the area of the middle lamella.

video moniton at a standard rate of 30 framesper second, 525 lines per frame. Vid«> outputfrom the photomultiplier was viewed on an 11-inch monitor (F), and hard-oopy micrographs(G) could be produced by photographing themonitor. It was also possible to make vid«>tape (H) for subsequent display on a secondmonitor (I I. Sequential photographs orphotographs of single video frames oould bemade in this operational mode (J).

The vid«> image on the monitors containedoonsiderable noise, but the effect wasminimized by using film expOSUR times ofabout 0.5 second. For RasoDS to be diacuseedlater, the image was defocused at the monitorso that individual scan lines blended anddisappeared.

While numerous Rfinements are possible,the equipment proved satisfactory for a varietyof dynamic experim_ts. Two examples will begiven heR.

When wood is subjected to large com-pressive forces perpendicular to the graindinction, tracheid walla bend or distort

Figure 2. - Substage to obeerveinelastic wood failure in com.pression.

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Figure 3. - Transverse surface ofsouthern pine progressively stressedin inelastic compressron per-pendicular to the grain.

As a second example, previous reeearchon the process for making groundwood in adouble-disk refiner (McMillin 1969) had in.dicated that individual tracheids of southernpine may fail while under torsional stress andunwind into ribbonlike elements. Suchelements provide the coherence necessary forstrength in groundwood pulps (Forgacs 1963).

motion is provided by a miniature 50-to-1speed reducer (A) with an anvil attached to theoutput shaft (B). The reducer is powered by ashaft (C) attached to a variable-speed DCmotor outside the specimm chamber. Thesurface of the anvil forms a plane passingthrough the axis of rotation. A stationary anvilis provided (D); its surface also forms a planepassing through the axis of rotation. The axialseparation is varied by adjusting themicrometer (E). Fibers are attached to theanvils with silver paint and rotated in the

Figure 4 is a detailed view of the mi-cromanipulative substage designed to applytorsional stresses to single fibers. Rotary

jo'igu/'e 4. - Micromanipulativesu6stage for stressing individualtracheida in torsion.

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clockwise direction at a rate of 40 degrees perminute.

Figure 5 illustrates the progressive for-mation of the ribbonlike element. MicrographA shows the undeformed fiber prior to ap-plication of torsional stress. As the fiber isrotated, stresS88 in the cell wall increase. Acritical point is eventually reached where thestrength of the fiber is exceeded and a crackabruptly forms at the zone of weakn88sdelineated by the fibril helix (point 1 ofmicrograph B). With additional rotation,further unwinding occurs by a tearing processin a direction generally following the fibrilangle. The areas between points 1 and 2 ofmicrograph C and between points 2 and 3 ofmicrograph D illustrate two steps of theproceaa. The original crack remains visible atpoint 1 in all micrographs of the series.

Digital Image Proee88iagSigDa. DigitiziDg

Before the image-processing capabilities ofthe digital computer can be applied tc faat-ecanmicroscopy. it is DeCessary to CODvert thesigDal (or displayed image) tc digital form.This can be doDe by various means. Assume

Figur. 6. - Deve~pment of a n"bbonlike elementin an earlywood traclieid of southern pine. The scalemark in A shows 6Gp.m. and is aleo applicable to B.C. and D.

that the sample 18 being 8C8nned at standardtelevision rates and that the image is displayedon a monitor. The problem then is to convertthis sigDa! to the slower rates required fordigital ~rding. The three most likelypossibilities are: 1) photograph the monioorwith a film camera and scan the film with someform of microdensitometer; 2) record the signalon vid~ tape for subsequent playback anddigitizing; and 3) digitize the video signaldirectly from the photomultiplier.

In the monitor-film-densitometer method,television monitors (unlees specially modified)produce a brightness which is nonlinearwith respect to the incoming signal. Therelationship betwMD film density and logexposure also is noDliDear. Theee two effectsmake it difficult to estimate the original signallevel from density measurements of the film.

Sharpness of image edges in the verticaldirection is optimum if the monitor 18 adjusted80 that the scan lines just blend and disappear.Any residual scan lines will produce a sev.-ebeat pattern with the digitizing raster unlessthe latter can be exactly superimposed on theformer, a practical impossibility. On the otherhand, if the scan lines are made to overlapexcessively, the image sharpness will bedegraded. In spite of its deficiencies, thissystem is the principal one in use. It requires aminimum of special equipment, and is low incost and simple to operate.

The second approach, recording the vid~signal on tape, allows replay for digitizing butadds considerable noise. Playback introducesfurther probl8Ds, since the normal vid~bandwidth necessitates a digitizing rate far inexcess of that which can be handled by con-ventional digital systems. Any additionalprocessing to obtain slower spMds (i.e.,transcription to a longitudinal recorder) addseven more no18e and may create time-baseinstabilities and jitter in the picture.

Memories capable of digitizing the fullbandwidth and reading out the signal at digitalrecording rates are available but very ex-pensive. Further, they generally can be used torecord only isolated frames 80 that extensivetape rewinding and frame finding would berequired to digitize a series of frames.Rotating-disk digital memories are becomingavailable and will provide multiple framestorage at more intermediate cost.

The third suggested method, directdigitizing from the video signal, is feasible only~

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when the image is repetitive. The techniquenormally is to digitize one column of pictureelements of each video frame, 80 that the timeto record a oomplete picture require8 a numberof frames equal to the number of oolumn8de8ired. If the dynamics of the 8ituationpermit, this method will produce the bestpicture quality. For standard television displayapproximately 17 s«ond8 would be needed forevery complce picture. Wken the sample i6moving at fa8ter speeds, the repetitive imagecan be obtained via a "frame grabber," adevice (normally a video disk recorder) such asis used in comm~l TV for atop-motiondisplay. However, signal-ta-noise ratio i8 againa problem.

Further oonsideration8 for hardware sys-tems applicable to the scanDing electronmicroscope are given by Billingsley (1971).Image Proee88lDg

Once the image is obtained in digital form,numerous type8 of processing are possible.Some applications, primarily in the aerospacefield, have been described by Nathan (1971)and Billing8ley (1970). In wood science, digitalprocessing of fast-scan picture8 can beillustrated with the micrographs of Figure S.

Figure SA was digitized via the monitor.film-densitometer route; the result is seen inFigure 6A. Some contrast was lost in scan.Ding-an effect that would not have occurredhad the signal been digitized directly from thephotomultiplier. The histogram displays therange of digital numb..8 represeDti. the gray

acale from black 00 white. The height of _chbar indicates the quantity of picture elementsat each digital number.

This image has large light and dark areas,each of which oontains detall whose k»caloontrast is small (by virtue of the restricteddigital range). Contrast stretching wouldimprove visibility of the detai18. Whilephotographic stretching would be useful in thelinear portion of the film-response curve, itwould cause brig~t areas to lose local contrastat the curve toe. The image may be stretchedby computer, but the saturation problemreappears when the output image isphotographically displayed. Therefore, afiltering method is used 00 remove the largedifference in brightness between the light anddark areas. The result of such filtering is shownin Figure 68. Again because of the limiteddigital range, the oontrast appears low. Now,however, it may be stretched without runninginto the toe and shoulder of the film-responsecurve when the output is displayed. Thestretched micrograph, f~ 6C, revealsconsiderable fine detail on' the cell walls andwithin the lumen cavities. Further, edgesharpness at the juncture of the cell wall andlumen is improved.

Digitized micrographs can a180 providequantitative data. As one illustration, Figure6A was partitioned at about 130 in the digital-numb. histogram. The effect was to producean image in which the lumen cavities and othervoids appeared black, while cell walls were

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JANUARY 1974276

Figur. 7. - A. Image resulting from partitioning Figure 6A at mid.gny. B. Differencepicture resulting by subtracting digitized versions of Figures 3 C and 3 D.

it is also applicable to light and tran8missionmicroscopy. Additional developments may beanticipated. It is the opinion of the authorsthat the microscopist of the near future will beprovided with a computer-driven displaysystem and will be capable of selecting, bykeyboard entry, a great variety of processingtechniques that will va8tly increase analyticalcapabilities and visual image modes.

white (Fig. 7A). A correlative computer listingof the partitioned digital numbers indicatedthat 47 percent of the micrograph was cell walland 53 percent was lumen cavity. Essentiallyidentical results were obtained when the areaswere planimetered.

In dynamic experiments, the motion whichoccurs between micrographs can be displayedby the digital computer through a processof image subtraction (Billingsley 1970).Micrographs 3A and 3B of the original com-pression sequence were digitized, manuallyregistered at the center row of cella, sub-tracted, and contrast-stretched. The resultappears in Figure 7B. The major deformationappears in the upper rows of cella as areas ofvery dark and very light tones, while areas oflittle or no change are mid-gray.

Literature Cited

BILL~G8L.Y, F. C. 1970. Applications of digitalimqe proce88ing. Applied Optk:1 9(2):289-299.

BILLlNG&8Y, F. C. 1971. Image processing forElectron Microecopy II: A digital 'f,tem. 111Advances in Optical and Electron Mlcro.copy.Vol. 4, pp. 127-159. New York; Academic Preas.

FO.GACI, O. L. 1963. The characterization ofmechankal pulps. Pulp and Peg. Mag. Can.64:T89-TI18.

McMn.LIN,C. W. 1969. AI~tI of fiber morphologyaffecting pro~rties of handaheetl made fromloblolly pine fermer groundwood. Wood Scl. andTech. 3:139-149.

NATHAN, R. 1971. Image proceuing for ElectronMicroscopy I; Enhancement procedures. 111Advances in Optkal and Electron Microscopy,Vol. 4, pp.85-125. New York: Academic Press.

Coacla8ioaThis paper has merely attempted to sketch

a few fast-sean EM techniques that offerpromise in wood science. While digital imageprocessing can augment fast-scan microseopy,

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