(7) Hahn, R. B., Baginski, E. S., Anal. Chim. Acta 14, 45 (1956). ( 8 ) Hahn, R. I+., IYeber, L., ANAL. CIIEM.28, 414 (1956). (9) Klingenherg, J. J., Pnpucci, R. A., Ibid., 24, 1861 (1952). (IO) Kumins, C. A,, Ihid., 19, 376 (1947).
(11) Menis, O., Oak
Ridge N a t i o n a l
Laboratory, Rcpt. ORNL 1626
(April 1954). (12) Oesper, R. E., I‘Xngenberg, J . J., ANAL.CHFM.21, 1509 (1!)49). (13) Thamer, B. J., Voigt, A. F., J . A m . Chern. Soc. 73, 3197 (1951).
J., VoiKt,, A. F., J . Phgs. C h r m 56, 227 (1!)52).
(14) Thnmcr, B.
RECEIVED for review October 8, 1956 Accepted December 26, 195K
Carbon Replication of Particulates and Ultramicroscopic Crystals JOHN H. 1. WATSON Physics Department, Edsel 6. Ford Institute for Medical Research, Henry Ford Hospital, Detroit 2, Mich.
b Single crystals and particles o f colloidal size can b e easily corbonmounted and carbon-replicated. Undissolved, such preparations of particle dispersions preserve the morphology of a complete distribution held rigidly within the relatively strong, structwreless mounting medium. Selective etching allows identification o f the components o f mixtures regardless of recognizable geometry. Carbon replicas of single crystals present improved opportunities for obtaining determinative crystallographic data in addition to mere morphology by electron microscopy o f a wide selection o f subsfances. Complicated microstructures can b e carbon-mounted, and b y a process of partial etching and stereoscopy, much con b e deduced concerning them. In interpreting the microstructures consideration must b e given to the monner in which the carbon deposits and to the effect that particle orientation has upon the electron image intensities.
C
replication (1) of surfaces for electron microscopy is a useful, convenient technique with wide application; it may be adapted to mount small particle dispersions for electron microscopy or to replicate the single tiny particles and crystals of such specimens. Finely divided powders have not been replicated extensively hy any process, although Calhick in particular has used silica replicas in the study of size and shape, of crystalline particulates in alkaline earth earhonate powders (34). Carbon replicas do not achieve results impossible with silica or othrr evaporated materials, although carbon does have some practical advantagese.g., greater resistance to chemical attack, apparently greater strength, and, mith respect to oxide replicas, greater electrical conductivity. Bradley (9)has apARRON
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ANALYTICAL CHEMISTRY
plied his carbon replica methods to iuvestigate the properties of single photographic grains in a manner similar to, but not identical vith, that reported here.
electron bomhardment of vaporizable materials, except that with carhonmounted pnrticlci: the rffrcts of hpat,
SPECIMEN PREPARATION
An aliquot of the sample, either in liquid suspension OT dry, is atomized or mounted from a dilute droplet upon a clean glass slide in a relat,ively thin deposit. The carbon is deposited vcrtically (Model SC-3, Optical Film Engineering, Inc., Philadelphia 33, f a . ) upon the slide and the resultant carbon film plus particulate sample is floated off on the surface of water or some other solution which dissolves the particles completely or etches out a given componcnt. Although there may be rlifficulties in etching the specimen properly, there is never any great difficulty in removing the carhon from the glass. It is not necessary to use a parting layer of any kind; after “squaring” the slide with a sharp object, immersion in the etchant frees the films. A little teasing is often necessary, and sometimes the films are left in the etchant overnight. If the specimen is not dissolved away from the carbon film it appears 11s a usual dispersion of particles. This is a distinct advantage over ordinary filmsupported samplcs in that the particles are locked in rigidly within the carhon film, which is not easily broken by the electron beam and can be relatively controlled in thickness. Silica films could he used similarly, although the preparation procedures would be different. Ah important consideration for those interested in the effects of heat, electrons, OT other radiation upon the specimens themselves is this: I n cases where the particles are affected by such radiations, the original size, shape, and surface characteristics of the particles are recorded in the carbon film. Such preparations are useful for studying the effects, where a single micrograph can record both the before and after aspects. This is similar to the process of preparing contaniination layer ghosts (If) by
.
Figure 1. Electron micrographs o f mixture o f carbon black particles and sodium chloride crystals mounted by carbon film A, 8. C.
X85,OOO X15.000
radiation, and t,hc likc outsirk the microscope can he similarly examined. Also, the vhole original morphology is recorded hy carhon-mounting, d,ereas hy a process of contaminatinn, certain phases m:by be srlectively lost, before or during csamination in t,hc microscope. Carbon-mounting would he uscful on a wider selection of substances t,han contamination layers, which tend to form more consistently on some materials than on otlrrm. INTERPRETATiON OF UNSHADOWED REPLICAS
Asimpleevample is shoanin Figure 1, C, of an artificial mixture of a carbon black and sodium chloride crystals carbon-mounted and floated upon water as a solvent for the salt. The mater has selectively etched the salt t o leave the carbon black imaffcctetl. The figure emphasizes the potent,ial uses of selective etching (or clifferent,inlsolution) of carbon-mounted pnrticulatcs in samples where a mixtiire of substances is SIRpected. Khen there is no recognizable morphology to assist in their idmtification, the elect,ron microscopy of mixtures is unrennrding; but tjy selcctively removing one or more compiments, the fractions of a mixtiire c:in he studied by convenient carbon-mounting or silicamounting techniques. By selectively etching carhon-mounted. p:rrticulate mixtures, one can also direct selected area diffraction more intelligently upon them, even in the microanalysis of tiny component parts of a single particle. Carbon replicas of single crystals present improved opportunities for securing determinative crystallo~raphic properties by electron micruscopy. If the crystals are boo large, ns tlie calcite crystal in Figure 2, the? tend to collapse and are of no value in determination of the morphology. If they arc of a smaller suitable size, the replicas oisingle crystals may show the crystnl faces, slight differences in their inclinations,
and often their srirf:m strnctnrcs. The observable differenccs in contrast hetween differently oricnted fnces is shown at higher magnification in Figure I, B. Carbon replication of single crystals allows an observer to see through them and to ohserve the arrangement of all the faces, because all faces except the one on rhich thr. crystal is snpported are replicated by the carhon. The d e positing carbon diffuses around the corners and deposits upon all surfaces to some extent, vitli t,he heaviest deposit on those faces t o m r d t,he carhon source. Calhick has disciisseerl the diffusion properties of silica and its failure to creep under a partirlc in similar studies ( S , 4 ) . Carbon follows suhstantiallg the behavior of silica (2). Figure 3, A , is an eler%ronmicrograph of a single crysbal of st,at:iconia (aragonite) taken from the static hhyrint,h of n frog. A drawing of bhc crystal (Figire 3, B ) indicates that it,is a slighdly imperfect orthorhomb. Figiirc 3, C, s h o w the normal ort~horh~rml~ for comparison. Severd anomnloiis situations may arise in t,he interpretation of such an image. For instance, in t,his micrograph, %s in Figure 1, A , and in mnny of bhc images of crystals in Figure 1, C, an area is observed over the crystal where the intensity is tlie s:ime :IS t,hnt of t,he surrounding film area and is considerably higher than that of the othcr fares of t,he crystnl itself. Thus, the cryst:il replica map appear to have sides with no t(ip or hottom siirf:iccs. This appearance is a result of two effcct,s. The first involves the fact that the carbon r:rpor, diffusing arorinrl the crystal edge and depositing on all surfaces, apparently is like silica in being unable t,o penetrnte beyond a certain boundary. Consequently, an area is left without carhon where the object rests upon the supporting glass slide. When the specimen is dissolved away, a holc
B
Figure 3. A.
is left in the bottom of the cryst,:il replica which manifests itself as an nrcn of higlielectron inknsity in the image or as a light area on the positive print. I n Figure 3, B, the crosshatched area represents this plane upon which the crystal was .supported and where there is no carbon. This is a five-sided planar area in Figure 3, B, but can take a variety of confusing shapes drpending upon the shape of the ohject and its orientation on the original slide. Thus in Figure 1, C, many xpare, light areas are secn; in Figure 1, B , where the crystal was balanced on a corner, the area is a three-pronged star; in Figlire 4 (a spherulite of calcite) a rounded ohject is ohsrrved to have a roiindcd light area over its imnge. This figure sliows t h t faces are heginning to form
Figure 2. Electron micrograph of carbon replica of relatively large, single cubic crystal of calcite, partiolly collapsed x10.000
C
Single orthorhombic crystal
Aragonite from slotaconio from static labyrinth of frog; X11.000
8. Plane$ of slightly imperfect orthorhomb shown in A. Crashotching a t x indicates plane of crystal which focsd corbon I O Y I C ~ and is oriented perpendicular to electron beom. C r m l o b h i n g a t y indicotes plane which supported crysld on slide during carbon evaporalion. Doubly croshotched area reprsrents clear area in A. C. Normal orthorhombic ~ r y s t o of l aragonite VOL. 29, N O . 4, APRIL 1957
563
on the sphenditrs :%s t,hey develop toward cubic h:hit. An electron micrograph of a replicated oval stataconium is shown in Fikure 5, A , whcre the film lins lxoken and the replica has rotated. The replica is observed from tlie side t,o shoa. the hole in its base directly. Illanre 5, B , C, and D,illustrates the mechanism whereby this hole has been formed The second cffect enters when all or part of the ripper surface of thc replicated crystal is perpendicular t,o the
incident elect,ron he:ini. Such an nren will have the same rehtively lox electron srat,tcring power :is tlie siirroiinrling film and xvill nppenr transparent compared with the inclined planes of the sides. A p r o j e t k l tlriikncss acts to increase effert,ivp clrdron s m planes a t angles bo t,he incident hcim have greater opacity for it, appear darker in t,he positive prints, and are, thercfore, ~list,ingnislinl,le. In t,lie rrpst a l in Figure 3, l j , thr rrossh:itrlred area x repnsent,s x top facr n-liich W:IS perpendicular to the clertron henm. Superposition of areas x and ?/ results i n a “ ~ l e a r ”nrea with the snnie intensity as the surrniinding filni. S.iperirnpasrrl on both are part,* of mrimrs other inclined,
cnrhoii-coatcrl siirf:%c?s. A sccond upper snrf:we nt IL slight inclination t,o the beam appeal.; rliglitly h k e r in the mirrograplr. Thnt t,he clear arr:is of siicli microg r q h actually hare film over them is dcrnonstratcd in Figures 6 and 7, vhere second smaller crystals, supported on thr clear a n n of the larger one, throw shxlorr-s upon it and in one rase beyond it. In order to interpret such images without amhiguity, the micrographs are t&n stcreoscopicnlly (Figure 7) and ohserved so that the base hole is affay from the vie\!-er. A third anomaly is thc well-known orcrirrence of light shaclo\\-s, 17.hicli are oft,en srcn stretching in :i, variety of dimct,ions from the rdges of carhonmounted particles in the positive prints. These nre a net result of the use of a relnbively large sourcc of carhon (0.6 cm.) xith short depositing distance ( I 5 cm.). Because of diffusion and depositinn of carlion intri the nrens, these shadows do not conPtitiitc holes in the fihii. Continiious, extremely thin earbon films \diicli scatter Plectrons t,o a negligible extcnt (.:in app:irently form and be mnintnincrl at least mit,liin the restricted arms of siieli shadom. APPLICATION TO COMPLICATED .STRUCTURES
Figure 4. Electron micrograph of carbon replicas of spherulites of calcite developing toward cubic habit X8000
Figure 6. Electron micrograph of carbon replica of stataconia (aragonite)
.
Smoller wy$tal is supported on clear orea of larger cryftol 10 01 to throw m shadow upon it and surrounding film; X8030
PARTICLE
-a
It i s practical to use mrhon replication wit,h t,iny three-dimensional objects of cornplictited, uIbr:%microscopic fine structure, particularly if the strmple is only part,i:ill? etched and examined stereoscopicdly (6). An example is the irsc of t,hc tcchniqoe wit,li powders of :iIpha.-iron dendrites (7-3). These arc of tlie order of a fraction of 1 to 10 mir:rons long, and contnin single crystal structures in t,he range of 50 to 500 A. arranged in an int,rirate, three-dirnensiond army. These complicat~edstmctures were msil>- carbon-moimtcd and pnrtinlly etched vit,h 4- to 6.V hydrochloric acid. Ry n m o n of tlie partial
. GLASS 1,
fi/ ///
I
/// qEPLICA I
Figure 5. Oval stataconium gonite), carbon replicated A.
(ara-
Carbon Rlm h o i been broken and curled over IO that replica i s seen from side. Hole in b a l e is shown; X9000 8, C. D. Origin of hole
564
ANALYTICAL CHEMISTRY
,.e
,. Figure 7. Stereoscopic pair of electron micrographs of carbon replicas of crystals of aragonite from stataconia of frog X6000
Figure 8. Electron micrograph of carbon replica of dendrites of alpha-iron partially etched with hydrochloric acid x50.000
Figure 9. Electron micrograph of dendrites of olpho-iron well etched with hydrochloride acid x50.000
nature of the rtching, dhose 1x2 :hes and regions in proximity ta t,he c ant dissolved most effrctively and me parts farthest from thc acid m r e relnt,ively iinnffecterl. In addition, t,he incomplete ctch left a skeleton framework o f the clenrlrites. Deposited in :I usrial manner upon n soplmrting film, the rvhole dendritr w s opnque and even stereoscopy of siicli s:imples did not yield a.n act:urnt,e intcrpretntion of microstructures. Mountr
