Electron Microscopical Studies of Natural Cellulose Fibers WILLIAM G. KINSINGER AND CHARLES W. HOCIC Hercules Powder Company, Wilmington 99, Del. Three developments i n the technique of preparing specimens for electron microscopy-metallic shadow casting, surface replicas, and electron stains-were applied t o a study of the submicroscopic structure of natural cellulose fibers. By metallic shadow casting, fine fibrils whose diarfieter varied from about 90 to 400 depending on the sample, were demonstrated in beaten liatural cellulose fibers. Larger fibrils, such as are observed in the light microscope and i n electron micrographs of unshadowed specimens, are composed of bundles of these finer units. The existence of these fibrils i n unbeaten fibers was confirmed by the examination of surface replicas in which contrast had been increased by metallic shadow casting. 1’2riodic variations in structure at about 150 along the long axis of the fibrils were revealed by shadow casting and by electron staining.
d.,
preparative techniques used for obtaining specimens thin enough to permit examination with the electron microscope, and perhaps also t o innate differences among samples. Most of the previous investigators employed the standard techniques of bright field electron microscopy in examining their specimens. I n the work described here, preliminary attempts have been made t o apply three recent developments in the technique of specimen preparation for electron microscopy t o a study of the submicroscopic structure of natural cellulose fibers. These techniques are metallic shadow casting, surface replicas, and electron stains. CONTRAST IN ELECTRON MlCROSCOPICAL IMAGES
i.
HE structure of cellulose fibers in the region below that resolvable with the light microscope and above t h a t adaptable to investigation by x-ray diffraction-Le., approximately in the range between 50 and 5000 A.-can be studied advantageously with the electron microscope. By means of this instrument a number of investigators have obtained significant information concerning submicroscopic fiber structure. Although their results frequently differ in detail, there is general agreement t h a t natural cellulose fibers have a fibrillate structurei.e., they are composed of fine filaments of cellulose called fibrils. Ruska and Kretchmer (29) observed fine fibrils on the order of 50 1.in cotton degradedoby hydrochloric acid. Kuhn (20) described fibrils 100 to 200 A. thick in cottoh fibers that had been treated with cuprammonium solution. I n samples t h a t had been ground, Hess, Kiessig, gnd Gundermann ( 1 2 ) observed fibrils ranging from 100 to 750 A. in diameter, whereas following chemical degradation ofocellulose fibers, Eisenhut and Kuhn (7) detected fibrils 100 A. wide. According to Wergin (34, 36), the fiber wall consists of elementary fibrils 80 to 100 A. in diameter, and these may occur in bundles. Sears and Kregel (30)found that mechanically disintegrated wood fibers were composed of “subfibrils” too fine to be seen with the light microscope, but easily detectable with the electron microscope. Sears and Kregel also reported work of Berkley and Greathouse, in which ground irradiated cotton fibers were observed to consist of rodlike pa;ticles, separate and in bundles, having diameters of 150 to 200 A. and lengths of 10,000 to 15,000 A. From chemically degraded cellulose Husemann (16) obtained electron micrographs of fibrils ranging down to BO A. in diameter. By using supersonics in the preparation of fiber samples for electron microscopical studies, Wuhrmann, Heuberger, and M ihlenthaler (87’) and Frey-Wyssling and Mohlenthaler (8) detected fibrils of various dimensions down t o 60 A. in diameter. I n a recent study, Hermans (IO) observed, in fibers that had been disintegrated by wet grinding, the general phenomenon of disintegration into fibrils whose diameters ranged down to 100 A. Similarly the work of Jentgen (181, Wallner (SS), Barnes and Burton (6),and others adds to the evidence in support of a fundamentally fibrillate structure for natural cellulose. Differences in the dimensions ascribed t o the fine fibrils by various investigators is probably due in large measure to different
It is becoming increasingly evident t h a t the limitation in the structural detail x-hich can be obtained in electron microscopical images is as often due t o the lack of sufficient contrast between the structural components of the specimen as it is to the resolving power of the instrument. Aside from instrumental factors, contrast is the result of the varying degree t o which different parts of the specimen scatter portions of the electron beam a t SO wide a n angle t h a t they are not grasped by the objective and do not reach the final image. Such scattering is directly proportional t o the thickness of the component, its density, and the atomic weight of the elements of which i t is composed. Lack of contrast is particularly noticeable in organic niaterials such as cellulose. The atoms of low atomic weight of which cellulose is composed-carbon, hydrogen, and oxygen-are poor electron scatterers. Therefore it requires a considerablo difference in either the density or thickness of contiguous structures to obtain photographically visible contrast between them. Such differences usually are not present; the density generally is of the same order of magnitude and differences in thickness are necessarily small for structural components of a size adaptable to electron microscopical study. As a result i t is frequently impossible t o discern details of structure in organic substances even when those details are of a size considerably larger than the resolving power of the microscope. The particles may be SO fine as t o have no contrast against their background, or structures within the boundaries of a large mass of material may have so little contrast among themselves t h a t the mass is seen only in silhouette. The result of low contrast in cotton linters fibers, which were dispersed by beating in water in a Waring Blendor and then mounted on a nitrocellulose film, is shown in Figure 1. The finer filaments show so little contrast against the supporting membrane as t o be barely visible. The larger filaments can be seen in outline but little or no internal structure is apparent. There is no way of ascertaining from such a micrograph whether the larger filaments are homogeneous or whether they consist of bundles of the smaller filaments. Fourtunately, there are mcthods by which the contrast of such specimens can be increased-namely, metallic shadow casting, as reported by Williams and Wyckoff (36), and electron stains, as reported by Hall, Jakus, and Schmitt (9). 1’711
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by the specimen or its substrate. IIow-ever, small variations in surface elevation of the specimen cause sufficient variation in the thickness of inet,al deposited so that they can be seen in thc image. I n ordcr i o obtain pictures in which the shadows were dark, prints were made from positive transparencies which, in turn, had been made froin the origina! negatives. I n photographs of t,his sort (Figures 3 t o 8), the specimen appears to be illuminated with light a t a n oblique angle. KOadditional detail is revealed a's a result of photographic reversal, but, the illusion of three dimensions is created, and this is helpful in interpreting the pictures and pleasing t o the eye. The pictures should not, be confused vr-ith true three-dimensional pictures obtained by steieoscopic electron microscopy.
Figure 1. Beaten Cotton Linters Mounted on Nitrocellulose Film, Unshadowed, 13,500 X METALLIC SHADOW CASTING
In metallic shadow casting, a metal of high atomic weight is evaporated onto the surface of the electron microscope specimen mounted on a supporting film. The metal must be one which shows no structure of its own in the election microscope when present in thin layers, 100 A. or less in thickness. Chromium in 80 A. thickness or gold in 8 3. thickness was recommended by Williams and Wyckoff (36). In a high vacuum the atoms of evaporated metal vapor travel in straight lines radiating from their source. If the source of the metal is small, and if the plane of the specimen is placed so that a beam of metallic vapor strikes it a t an oblique angle, elevated points on the surface of the specimen prevent the metal from depositing upon lower areas t o the leeward. I n the electron microscope the metal-free areas or "metallic shadows ' are more transparent t o the eiectron beam, and the image appears as shown in Figure 2. I n such a specimen the scattering of the electron beam by the metallic film considerably exceeds the scattering caused
Figure 2.
Positive Print of Chromium-Shadow-ed Linters, 16,000 X
Cpmmercially purified staple cotton, cotton linters fibers, ramie, and viood pulp viere examined by this t,echnique. (As used in this paper, linrers fibers refers t'o the short fuzz hairs, whereas staple cotton refers to the longer lint.) Because individual fibers are too thick for use in the electron microscope, they were mechanically disint,egrated by beating in water in a Waring Blendor for 15 to 20 minutes. The aqueous dispersion rvas then allo\r-ed to sct,tle slightly, whereupon small drops of the supernataiit siurry were placed on a glass slide which previously had been coated with a thin film of nitrocellulose (0.37, Parlodion in amyl acetate). After the water evaporated, leaving the finely dispersed cellulose on the supporting film, the specimen was shadowed eit,her with chromium or gold. Many experiments of this typo indicated that chromium is to be preferred to gold for reasons of easier experimental technique and lack of artifacts in the evaporated films. An angle of 45" or higher was found to be preferable to thelow (15") angles suggested previously (36). The greater angle v a s especially advantageous for revealing internal details, as in a large bundle of fibrils. After shadowing, the film was floated off the glass slide onto water and prepared for examiiiation in the electron microscope by the usual methods. S o objective aperture was used in the microscope with which these micrographs were obtained. Figures 4,.5 , 6, and 7 are photographica'ly reversed prints of micrographs obtained from specimens shadoffed w-ith chromium, and Figure 8 is a similar print of a specimen shadowed with gold. In all the micrographs it is apparent that' the specimens are composed of fibrils. The coarser fibrils are seen not t o be homogeneous but t o consist of still finer ones. The diameters of several hundred of these fine fibrils from each of the various fiber samples were measured by dividing t,he distance across several contiguous fibrils by the number of fibrils which were traversed, \k-ithin the limitations of technique, the diameter of the finest' fibrils appeared to vary in any one sample. Their approximate average diameter !vas as follows: ramie, 370 -1.;cotton linters fibers, 160 A.; staple cotton, 100 b.;
Figure 3.
Negative Print of Figure 2, 17,000 X
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Figure 4. A. Beaten Cotton Linters Shadowed with Chromium at 45' Angle, 26,000 X. B . Photographic Enlargement of Same Specimen but Different Field of A , 120,000 x
Figure 5. A. Beaten Cotton Staple Shadowed with Chromium at 45" Angle, 21,000 X. B . Photographic Enlargement of Portion o f A , 111,000 X
and wood, pulp, 90 A. These values cannot be considered absolute, but only indicative of the relative coarseness of the fibrils in the specimens under the conditions of examination. The variations of fibril diameter in any one specimen, the thickness of the metallic film, and the angle of shadowing-all would affect the relation of the above values t o an absolute or true fibril diameter. However, in the preparation of the specimens, the last two variables were maintained as constant as experimentally possible. From the micrographs it is not evident whether the fine fibrils are aggregated into units of a larger size, such as the fibrils several tenths of a micron in diameter which have been reported frequently from studies with the light mhroscope (1-4, 18, 13, 16, 27,28, $ 1 ) . Although bundles near this size range can be found, larger and smaller groups are also present. It is likely, of course, that the beating process would disperse any larger units that may exist. I n some of the samples, particularly in cotton, the fine
fibrils seemed to be arranged in layers. Measurements of the length of the shadows cast by these layers, when corrected by the known angle of shadowing, showed them to be on the order of 0.1 micron in thickness. This figure is in satisfactory agreement with the thickness reported for the growth rings in cotton ( 1 4 , 1 9 ) . iln interesting structure was detected in metal-shadoned fibrils upon examination a t high magnifications. As shown in Figures 4, 5, and 8, transverse markings can be found more or less evenly spaced along the length of a fibril. The distance between these markings, whose arrangement occasionally suggests a spiral configuration, was about 150 A. It has not yet been possible to determine the cause of such markings. They could be due t o coalescence of the metal film into evenly spaced droplets. That this phenomenon does sometimes occur is generally recognized by investigators who have used this technique. Hoir ever, no such behavior in chromium-shadowed specimens has been noted by the authors or reported by others. The possibility
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Figure 6.
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Beaten Ramie Shadowed with Chromium at 45’ Angle, 16,000 X
Figure 7. Chemical Wood Pulp Shadowed with Chromium a t 45 O Angle, 19,000 X
Figure 8. A . Beaten Cotton Linters Shadowed with Gold at, 4 5 ’ Angle, 40,000 X. B . Photographic Enlargement of Portion of A , 88,000 X
that this pattern in some way reflects an internal micellar or crystalline structure in the fibril is of interest, and warrants furthcr study.
what a i first glance looks like a surface elevation later appears to be a depression. Only by careful study and by recognit,ion of the original direction of shadowing can the true character of the specimen be deduced. At any rate the pattern of surface markings shon-n by the replica clearly reflects the fibrillate nature of the fiber cellulose. The smallest fibrils outlined iii the replica have about the same diameter as t,hose observed in fibers which had been disintegrated mechanically and then shadowed with met,als. At more or less regular intervals, deep crevices approximately parallel t o the long axis of the fine fibrils occur in the replica. The dist,ance between these crevices is of the same order of magnitude as the coarser fibrils frequently observed with the light microscope; this suggests that the finest fibrils may be aggregated in bundles.
SURFACE REPLICAS
I n order to study the surface character of the fibers used in this study and to obtain information about the fibrillate pattern of unbeaten fibers, metal-shadowed replicas were examined. A thin film of nitrocellulose was cast from solution (3 OVo Parlodion in amyl acetate) on a microscope slide. Before the film was completely dry, several individual unbeaten fibers were pressed gently against it. When the solvent had evapoiated, the fibers were stripped off with tweezers, leaving their surface imprint in the film, which was then shadowed with chromium a t an angle of 15’. Subsequently, the films were removed from the slides and examined in the electron microscope. A typical replica is illustrated in Figure 9. In examining this photograph one should bear in mind that he is viewing a replica and not the original fiber surface. It has been noticed that pictures of this sort often “invert” while being viewed, so that
ELECTRON STAINS
If compounds containing atoms of high atomic veight can be attached select,ively t o parts of an object,, contrast between the parts is increased, and there is aeconiplished for electron microscopy what is comparable t o differential staining of objects for
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Figure 10. Beaten Cotton Linters Stained with Lead Acetate. A . 28,000 X. B. 73,000 X
Figure 9. Nitrocellulose Replica of Surface of Cotton Linters Fiber, Shadowed with Chromium at 15” Angle. A . 19,000 X. B . 50,000 X
examination with visible light. After treatment with reagents of high electron-scattering power, certain biological structures have been studied successfully with the electron microscope. Muscle tissue ( 9 )and bacteria ( d l ) ,for example, have been shown to take up, selectively, certain solutions containing heavy atoms, so that subsequent examination with the electron microscope reveals details of structure not otherwise detectable. By treating molluscan muscle fibrils with phosphotungstic acid, Hall, Jakus, and Schmitt ( 9 ) revealed a regular geometrical pattern characterized by cross striations spaced about 145 A. apart. With the idea that a similar method might be applied t o cellulose fibers so as t o reveal details of their structure, particularly differences between amorphous and crystalline regions, commercially purified cotton linters fibers were treated with a variety of solutions containing heavy atoms. The fibers were broken down into fibrils by beating in a Waring Blendor. A slurry of the beaten fibers was treated with the
“stain,” and then washed by centrifuging several times with distilled water. A drop of the suspension was placed on the specimen screen, either with or without a nitrocellulose film for s u p pqrt, and allowed to dry before examination with the electron microscope. Because these experiments were of a n exploratory nature, various concentrations of the staining reagents, ranging from 1 to 20010, were used. The duration of contact with the stain ranged from several minutes to several weeks, the average being about 20 to 30 hours. Nost of the reagents were applied a t room temperature, but in a few cases the fibers were treated at 0 ” t o 4 ” C. A ueous solutions of the following reagents were used: mercuric chloride, lead acetate, silver nitrate, copper sulfate, uranyl acetate, thallium hydroxide, phosphotungstic acid, 0.3% iodine1.5% potassium iodide, and zinc chloride-iodine (Herzberg’s stain). A half-saturated solution of mercury-potassium iodide in glacial acid was also used and applied to fibers which had been beaten in glacial acetic acid instead of water. The most satisfactory results, as evidenced by darkened areas within the celiulose framework, were achieved with aolutions of lead acetate. Although variations were encountered among samples that had received similar treatments, i t seemed significant that in most cases the darkened areas occurred not randomly but in a more or less definite pattern, frequently as bands running approximately transversely t o the axis of the fibrils. I n Figure 10, which is a micrograph of a specimen t h a t was treated for 18 hours with a 20% aqueous solution of lead acetate at room temperature, the distance between these bands is on the order of magnitude of 100 t o 200 1. This spacing is in
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reasonable agreement with that measured in cotton after chromium shadowing. SUMMARY
The results of this investigation offer additional evidence in support of a fibrillak structure for natural cellulose. The coarse fibrils commonly observed by optical and electron microscopy are shown b metallic shadow casting to consist of still finer unit fibrils. h , h o u g h the diameters of these fine fibrils vary somewhat in s, single sample, greater differences in this respect .prevail among fibers of different origins. Thus, the diameters of the fibrils in ramie, cotton, and wood pulp decrease in the order named. Similarly, variations in straightness and smoothness of the fibrils follow the same order. These differences suggest a correlation between the character of the fibrils and the physical and chemical properties of the fibers. Ramie, for example, is a highly crystalline, well-oriented fiber wit,h marked tensile strength but Kith low elongation and poor reactivity. The propert'ies of lsood pulp fibers are in some respects opposite those of ramie, whereas cotton is, in general, intermediate. It the fibrils, iii contrast to the interfibrillar regions, are niore highly ordered, one would expect chcrnical reactions to proceed along the fibrillar surfaces before the fibrils themselves were att,acked. The volume of interfibrillar cellulose is, of course, inversely proportional to the diameter of the fibrils. Other things being equal, reactivity should then correlate with fibril diameter. Some basis for the postulate may be found, for example, in hydrolysis studies where t'he accessibilit'g to acid degradation is considered to be a measure of amorphous cellulose (6, 22--dS). It seems significant that the accessibility of ramie, cotton, and wood pulp correlates n-ith the magnitude of their fibrillar surface. Neither the,widths zf the fibrils nor the lengthwise periodicity of approximately 150 A. observed after chromium shadowing and after electron staining correspond t o the dimensions of submicroscopic structures determined by other methods. By x-r$,y diffraction, for example, the micelle is found t o be about 50 A . wide and a t least 500 A . long ( 3 2 ) . A4sestimatcd from the degree of polymerization of hydrolyzed ccllulose (26), the more highly ordered regionsjn cotton consist of 280 glucose units-Le., approximately 1450 A. long. Following hydrolytic and oxidative breakdoKn of cotton and ramie cellulose, Husemann and Carnap ( 1 7 ) found, in size distribution cyrves of the remaining fiber segments, a maximum a t 2250 * 250 A. All these figures must be resolved in any final picture of the sinall scale structure of cellulose. ACKNOW LEDGR.1EST
The authors are indebted t o A. N. Abbott for much of the experimental work involved in obtaining the micrographs.
LITERATURE CITED
(1) Anderson, D. B., and Kerr, T., IND. ENG.CHEX.,30, 48 (1938). (2) Bailey, I. W., "The Cell and Protoplasm," Lancaster, Pa., Science Press, 1940. (3) Bailey, I. W., and Berkeley, E. E., Am. J . Botawg, 29,231 (1942). (4) Balls, W. L., and Hancock, H. A,, Proc. Roy. SOC.( L o n d o n ) , B93,426 (1922). { 5 ) Barnes, R.B., andBurton, C. J., ISD.ENG. CHEM.,35, lZO(1943). (6) Conrad, C. C., and Scroggie, 4.G., Ibid., 37, 592 (1945). (7) Eisenhut, Q., and Kuhn, E., Angeto. Chem., 55, 198 (1942). (8) Frey-Wyssling, A., and MtMenthaler, K., Textile Research J . , 17, 32 (1947). (9) Hall, C. E., Jakus, M . A., and Scbmitt, F.O., J . Applied P h u s . , 16, 459 (1945). (10) Hermans, P. H., Textile Research J., 16, 545 (1946), '(11) Ress, K., Kiessig, H., and Gundermann, J., 2. physik. Chem., B-49,64 (1941). '(12) Hock, C . W., J . Research NatE. B u r . Standards, 29,41 (1942). '(13) Hock, C. W., Teztile Research J., 17, 423 (1947) (14) Hock, C. W., Ramsey, R. C., and Harris, M., J . Research iYatl. B u r . Standards, 26, 93 (1941). ((15) Hock, C. W., and Seifria,W., P a p e r Trade J., 110, T-63 (1939). ~(16)Husemann, E., J . makromol. Chem., 1, 16 (1943). '(17) Husemann,E., andcarnap,A., Naturwissenschaften, 32, 79( 1944). ((18) Jentgen, H., Runstseide, 23, 76 (1941). ,(19) Kerr, T., Protoplasma, 27, 229 (1937). ((20) Kuhn, E., Melliand Tertilber., 22, 249 (1941). (21) Mudd, S., and Anderson, T. F., J . E x p t l . X e d . , 76, 103 (1942). ((22) Nickerson, R. F., IND. EWG. CHEM.,33, 1022 (1941). (23) Ibid., 34, 1480 (1942). ((24) Nickerson, R. F., and Habrle, J.A., Ibid., 37, 1115 (1945). '(25) I b i d . , 39, 1507 (1947). ~(26)Philipp, H. J., Nelson, 131,L., and ZiiRe, H. M., Ttstile Research J., 17, 685 (1947). (27) Ritter, G. J., IKD. EXC.CHEM., 20, 941 (1928). (28) Rollins, M . L., Teztile Research J . , 15, 6 5 (1945). (29) Ruska, H., and Kretchmer, M . , Kolloid-Z., 93, 163 (1940). (30) Sears, G. R., and Kregel, E. A., P a p e r Trude J., 114, T-43(1942). (31) Seifriz, W., and Hock, C. W., Ibid., 102, T-250 (1936). (33) Simon, W.A , , in "Cellulose and Cellulose Derivat,ives," ed. by E. Qtt, New York, Interscience Publishers, 1943. (33) Wallner. L., Melliand Testilber., 23, 158 (1942). (34) Wergin, W., B i d . Zenl?., 63, 350 (1943). (35) Wergin, W., Kolloid-Z., 98,131 (1942). (36) Williams, R. C., and Wyckoff, R. W.G., J . A p p l i e d Phys., 17, 23 (1946). (37) Wuhrmann, K., Heuberger, A., and Mtlhlenthaler, K., Experientia, 2, 105 (1946). I
RECEIYED Xovember 13, 1947. Presented before the Division of Cellulose Chemistry a t the 110th k1eeting of the AMERICANC s E n i I c h L SOCIETY, Chicago, Ill.
BUTYL ALC From Xylose Saccharification Liquors from Corncobs A. F. LAiYGLYIZI
