The Electron Microscope and Cellulose
.
R. BOWLING BARNES AND CHARLES J. BURTON Stamford Research Laboratories, American Cyanamid Company, Stamford, Conri.
URING the past twenty or more years, numerous investigators have reported exhaustive studies relating to cellulose, ita structure, and its physical properties. For example, Balls and Hancock (6) concluded that cotton fibrils were 0.4 p in diameter. Hereog (11) reported a diameter of 0.3-0.5 p, and Frey-Wyssling (IO) found it to be 0.4 ,u, Other workers, such as Preudenberg (9), Bailey and Kerr (4),Anderson and Moore (g), and Anderson and Kerr (1) expressed the opinion that fibrils have no consistent size and grade down to the limits of microscopic resolution. Seifriz and Hock (17') reported the existence of primary fibrils of 1.4,~ diameter and of secondary fibrils of 0.1-0.3 p diameter. Farr and Eckerson (8) recognized the existence of ellipsoidal building units 1.1 X 1.5 p in dimensions. Recently Bailey and Brown (3) determined that the diameters of cellulose fibrils vary from 0.91 to 0.97 p, depending upon the source of material. Many other scientists have made studies in this field, but the above citations serve to show the wide divergence of views which have been presented. Thus far, no common denominator has been found for these investigations, although heated controversies have frequently occurred in print. It is not our intention to enter into these discussions. Our sole aim is to present a series of recently completed observations; the electron microscope has been used to photograph cellulose which has been mechanically disintegrated in water. At the same time a direct comparison is shown of photomicrographs and electron micrographs taken of exactly the same pieces of cellulose. It is believed that the pictures will be of considerable interest and help in connection with the proper interpretation of some of those published in the past by other authors. The electron microscope has been described in detail in many papers, both scientific and popular; hence no such discussion need be given a t this time. In a recent paper from this laboratory (6) a method was described whereby electronic magnifications as low as 160 diameters can readily be obtained without any appreciable loss of depth of focus or resolving power. These low magnificationa make possible a direct comparison a t the same magnification between optical and electron micrographs of the same specimen, and have proved of great value in a wide variety of electron microscopic studies. The use of any optical instrument for the study of objects so small that they are comparable to or smaller than the limit of resolving power of the instrument is complicated by many factors, Among these is the fact that the depth of focus of the high-resolving-power objectives is of necessity so small (down to 0.06 p for a theoretical resolving power of 0.15 p) that it is practically impossible to focus sharply a t any one time more than a few scattered particles out of a given field. It can be understood that, while these few particles aTill be portrayed by the objective as accurately as possible, all others, whether larger or smaller will, if they do not have their largest cross sections accurately located in this shallow region
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of sharp focus,give rise to images which are out of focus and greatly enlarged. Appreciable error3 will also arise from the unavoidable presence in the image of diffraction effects. Inasmuch as cellulose fibrils and crystallites or particles have been reported to have diameters ranging from 0.1 to 1.4 ,u, and since the limit of resolution of the best visible light microscopes is also of this same order of magnitude, we have believed that some of the chief characteristics of many published photomicrographs of cellulose have been determined by diffraction effects. The present study was therefore indicated, and the results show rather clearly that such has been the case.
Diffraction Effects and Optical Microscopes As a result of diffraction, each point in an object is portrayed by the objective lens as a disk surrounded by a set of faint rings, the diameter and appearance being a function of the wave length of light used and the geometry of the lens. The diameter of the central disk of the image is given by the expression: d =
2x
hA
(when object is illuminated by parallel light)
where h
= Rave length of light used S A = numerical aperture of lens
Attention must be called to a fact important for the proper interpretation of the results shown; namely, the diameter of the small object does not enter a t all into the expression giving the size of the diffraction image. It has been shown both theoretically and experimentally that these diffraction effects play a decisive role in limiting the resolving power of all optical instruments. Rayleigh pointed out that two very small objects can just be revealed aa a doublet when the centers of their images are far enough apart to cause the first diffraction minimum of one image to coincide with the center of the diffraction maximum of the other. Abbe shon.ed that the practical limit of resolving power for a microscope can be expressed by the formula: E = - =2n- hsin
h
(oblique illumination)
where X
= mave length of light, microns N A = numerical aperture of objective n = refractive index R = distance between centers of two small particles which are just resolved
The maximum value of N A for any lens system used in air is slightly less than unity. If oil immersion objectives are used, N A can be made nearly equal to 1.5. Consequently, the best resolution which can be obtained is given by the expression:
OF PHOTOMICROGRAPHS (left) AND ELECTRON MICROGRAPHS (right) OF THREEOF FIGURE 1. COMPARISON FIELDS OF CELLULOSE
THE
SAME
Both micrographs were originally made a t 1000 X b u t are enlarged here t o SO00 X. The photomiorogrsph was taken with green light, using a 4-mm. apochromatic objective, N A 0.95, depth of foona 0 8 , 0.08 p . In order to photograph t h e 8ame tield as t h a t shown in the electron miorogra h the sample waa allowed t o remain on the nitrocellulose film suspended over a mesh opening i n a metallic screen of 200 meeh per inch. T k e conditions used in elaotron micrography are not t h e optimum ones in photomicrography,, and judgment of t h e photomicrograph should be made with consideration of tho prevailing conditions. The intensities of the diffraction disks in the photomicrograph are proportional t o the slze of the diffraoting point as shown in the electron micrograph.
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will possess edges which are broadened by diffraction by an amount equal to R; this places a limit upon the accuracy with which the size of any given object may be determined. The above discussion of diffraction disks can be shown (7, 1.2, 18) t o hold, whether the small objects are themselves luminous, as in the case of stars, whether they are opaque particles, or whether they are transparent objects whose refractive index is different from that of the surrounding media. I n fact, these objects need only be optical inhomogeneities in order t o produce the diffraction phenomena referred to above. More complete treatments of this subject may be found in almost any standard textbook on optics. I n these books, illustrations of the diffraction effects present in the images of stars, slits, needles, round opaque disks, etc., produced by both telescopes and microscopes, may be seen. Detailed discussions are given of the Fraunhofer and the Fresnel types of diffraction, as well as the reasoning and experiments which led t o the above mentioned formula for resolving power. For purposes of subsequent discussion, it need only be stated here that any minute object capable of absorbing, scattering, or diffracting light will be portrayed by a lens as an image in which diffraction Rill play an important role. As the size of the particular piece of matter which thus disturbs the light waves incident upon it decreases, the effects of diffraction become of greater importance in the production of the image, until finally the image is entirely produced by diffraction. While the size of such an image may be shown to bear no direct relation to the actual dimensions of the original particle, it is true that its relative intensity is a function of the particle size. From this point of view, accordingly, a field made up of a plurality of small pieces of matter whose centers are separated by a distance equal to or less than R will not be resolved by the particular objective used in calculating R. If, on the other hand, the field consists of objects separated by distances larger than R and varying in size about the value of R, the resulting field will be made up of a multiplicity of rounded-off images of varying intensities. While the larger, more intense images seem to have some definite shape, the others are almost round. The latter images vary downward in size until a certain minimum diameter is reached, and their intensities decrease until the images of the smallest particles fail to stand out against the general background. Since the wave length associated with electrons, accelerated by a potential of 55,000 volts is, according to de Brogile, given by the expression
x FIGURE 2 ( T o p ) . EDGEO F UNFIBRILaTED 15-DAY COTTON FIBER( X 7500) ; LONGITUDINAL STRIATION Is APPARENT FIQTJRE 3 (CenffY). M.4TERIAL LEACHED OUT OR WASHED FROM THE SURFACE OF 1 5 - D A y COTTON FIBERS IN WATER SUSPEXSION ( X 7500) FIGURE4 (Bottom). PORTION OF COTTONFIBER WHICH HASSWELLED AND COMPLETELY LOSTITS IDENTITY AS A RESCLTOF INTENSE ELECTROX BOMBARDMENT ( X 7500)
For visible light of wave length 0.6 p, the smallest resolvable distance will, therefore, be approximately 0.2 p. By using ultraviolet light, this distance can be decreased by a factor of about 2. I n the case of single objects larger than this limit of resolution, the diffraction effects referred to above are superimposed upon the true refraction or absorption image. The resulting image, although conforming to the true shape of the object,
=
4%
x
10-4~,
and is equal to 0.0000052 p, the electron microscope should be capable of resolving particles many times smaller than those which can be studied successfully in the optical microscope. It is well known that this is true. Objects have been clearly resolved by the electron microscope whose centers are separated by less than 0.004 p. I n these images the magnitude of the diffraction effects is extremely small. Therefore, if identical pieces of matter are photographed by both optical and electron microscopes, it should be possible to illustrate and determine the extent to which images produced by the light microscope are made up of diffraction effects. This has been done, and the photographs of cellulose shown in Figure 1 warrant particularly close and detailed examination.
Application to Cellulose Problems Since electrons are absorbed and scattered by air, the electron microscope must of necessity be operated a t greatly
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be recorded since the swollen places were in a rapid state of motion. Sometimes they ran t o and fro along the fiber, and a t other times they swelled to many times the diameter of the original fiber and finally burst and collapsed. Once such a fiber had become thoroughly charred or burned, however, it would come to rest and further irradiation failed t o produce additional changes. Since experience has shown that thin sections must be of the order of 0.1 p if satisfactory electron micrographs are to be obtained, little help could be expected from attempts at cross sectioning. One available technique, however, for minimizing the heating effects consisted of reducing drastically the intensity of the incident electron beam. Such a reduction obviously made proper focusing difficult but yielded encouraging results. Figure 2 shows an electron micrograph made in this way. The most satisfactory results were finally obtained by subjecting the cotton fibers to mechanical disintegration prior to their introduction into the microscope. In this way the fiber fragments were reduced in diameter to a point where they were no longer opaque to the electrons. By constantly observing the large field visible in the intermediate image, it was easily possible to determine whether any changes in the appearance of the fragments took place as they were maneuvered into the electron beam. As a further check, optical photomicrographs were made of a given field both before and after the electron microgapha had been taken. In no case was any change in the appearance of such a finely comminuted specimen observed. I n making these studies, cotton fibers were taken directly from bolls, some 15 days old and others mature. These FIGURE 5. FIFTEEN-DAY COTTON FIBER( X 6000) PARTIALLY (above) AND fibers were then suspended in water MORECOMPLETELY DISINTEGRATED (below) and subjected to the disintegrating acTwo phaaes can be seen in each micrograph: a very dense material whiah is almost aompletely opaque t o eleotrons. and an extremely fibrous material. tion of a Waring Blendor for approximately 30 minutes. A drop of the supernatant liquid waa then allowed to dry on a nitrocellulose or Formvar holvvinvl formal) membrane mount, following the usual tec’inGue of reduced pressures. For best results a vacuum corresponding electron microscopy. to about mm. of mercury must be maintained. Thus, Figure 2 shows a portion of a whole fiber from a 15-day all specimens introduced into the microscope are strongly cotton boll. The sample was simply stirred in water, a suitdehydrated. Furthermore, if the beam of fast-moving electrons incident upon the specimen is suddenly stopped by able mount prepared, and the electron micrograph made at reduced electron beam current. Figure 3 was obtained from the object, an appreciable amount of heat is produced. I n view of these two effects, some doubt was entertained the same specimen mount as Figure 2. It shows material by the authors as to the applicability of thq electron microwhich has either been leached out of the fiber or washed from its surface. Figure 4 shows the effect of heat referred to scope to cellulose problems. A few preliminary attempts to examine whole cotton fibers confirmed our suspicions. Since above. The electron micrographs in Figures 5 and 6 are typical of a cotton fibers are of the order of 18 p in diameter, the heating great many fields which we have recorded. They show the effect was particularly bad. As soon as the mechanical stage was manipulated so as to bring a given fiber directly into the appearance of both 15-day and mature cotton fibers which electron beam, the image on the fluorescent soreen showed have been disintegrated in aqueous suspensions. The presence of two rather distinct phases, one fibrous and the other that the fiber underwent marked changes. Probably as a result of internal heating, the fiber would swell locally at apparently amorphous and extremely opaque to electrons, is several places. No satisfactory electron micrograph could clearly seen.
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Comparison with Photomicrographs Observations of these fields were made
at 500 diameters with an optical microscope. I n no cases did the optical images appear to be very similar to the electron images, which were usually observed on the fluorescent screen at about 6000 to 10,000 diameters. The reason for this apparent anomaly could not readily be ascertained, since it mas never possible to know with certainty that exactly the same pieces of matter were being observed in the two microscopes. Accordingly (6) experiments were conducted as a result of which it became possible to obtain electron micrographs at low magnifications. By comparing directly photomicrographs and electron micrographs, as in Figure 1, the explanation for the lack of similarity is immediately apparent. From these comparisons it appears that the conclusions expressed in the introduction of this paper are correct. It can be shown that: 1. The chief characteristics of theoptical image of these finely divided pieces of matter are a result of diffraction effects and the fact that many parts of the field are slightly out of focus. 2. Small pieces of either of the two phases discussed above give rise to images very similar in appearance. 3. An apparent minimum “particle” size does exist in the cases of inhomogeneities smaller than the limit of optical resolving power. 4. In the case of the diffraction images, the intensities vary and decrease as the size of the objects decrease. 5. The smallest objects shown by the electron microscope fail to shorn up in the photomicrographs. 6. It is impossible to estimate accurately the size or the shape of such small objects from a study of their optical images. 7. Many types of inhomogeneities, such as the crossing or branching of filaments whose diameters are far below the limits of resolving power of the visual microscope, are blown up by diffraction effects into rounded images which in some cases are many times too large. Similarly, minute objects or isolated bits of debris appear also as rounded and enlarged images.
I
FIGURE 6.
biATURE COTTON
BOLLFIBERSMECHANICALLY DIsINTEGRaTED
IN WATER
( x 6000)
Bundles of filamenta aTe seen to be stripped from the large fiber at t h e lower left of the top miorograph.
In order to broaden the scope of this work, samples of cellulosic material from several other sources were prepared in a similar manner. Figures 7, 8, and 9 show typical fields of Whatman’s grade 0 filter paper (mixture of cotton and wood cellulose), kraft pulp taken directly from a laboratory beater, and yellow pine sawdust. Apart from minor differences which can be explained by a consideration of the source of the material, all of our electron micrographs of cellulose shorn the same type of structure. Attempts have also been made to disintegrate cotton fibers in other than water suspension. Almost no mechanical d i s i n t e g r a t i o n was found for cotton
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However, several of our electron micrographs are of special interest because they show direat comparisons with optical photomicrographs. The comparisons have been made primarily as part of a larger program of work undertaken to study the limitationa of both optical and electron microscopy.
Literature Cited
FIGURE 7. FILTERPAPER(WHATMAN’S GRADE0) MECHANICALLY DISINTEGRATED IN
WATEIR( x soooj
FIGURE 8. KRAETPULPAFTER HAVINCI PASSED THROUGH LABORATORY BEATER( X 3250)
A
suspended in n-hexane, cyclohexane, benzene, and absolute alcohol, even when the disintegration was continued for long periods. Examination of the cotton after such treatments showed no signs of mechanical fibrilation.
Conclusions The work which we have briefly outlined has been done at various intervals during the past year. During that time, several papers, primarily of European origin, have been published on various phases of this same problem (13-18). Our results concur in general with those already published.
(1)Anderson, D. E., and Kerr, T., IND.EIQ, CHEM.,30, 48-54 (1938). (2) Anderson, D. E., and Moore, J. H., Am. J. B O ~ U W24, , 503-7 (1937). (3) Bailey, A. J., and Brown, R. M., IID. ENQ.CHeM., 32, 57-63 (1940). (4) Bailey, I. W.,and Kerr, T., J . Arrzold Arboretum, 16, 273-300 (1933). (6) Balls, W. L., and Hanoock, H. A,, PPOC. Roy. Soo. (London), B93,426-40 (1922). (6) Burton, C . J., Barnes, R. E., and Rochow, T. G., IND. ENQ. CHEIM., 34, 1429-36 (1942). (7) Drude, P., “Theory of Optics”, Kew York, Longmans, Green & Co., 1929. ( 8 ) Farr. W. K.. and Eckerson. S. H.. Contrib. Boyce Thompson Inst., 6 , 189-203 (1934).
FIGURE 9. YELLOW PINESAWDUST MECHANICALLY DISINTEORATED IN WATER (x 3250)
(9) Freudenberg, K.,J . Chem. Education, 9,1171 (1932). (10) Frey-Wyssling, A., “Die Stoffausscheidung der hoheren Pflanzen”, 1935. (11) Herzog, R. O.,Papier-Fabr., 23, 121-2 (1925). (12) Jenkins, F. A.,and White, H. E., “Fundamentals of Physiaal Optics”, New York, McGraw-Hill Book Co., 1937. (13) Kuhn, E., Melliand Tmtilber., 22,249 (1941). (14) Lundgren, E. H.,Tek. Tid., 71, Uppl. A-C, Kemi 29 (1941). (15) Ruska, H.,and Kretschmer, M., Kolloid-Z., 93, 163 (1940). (16) Sears, G. R., and Kregel, E . A., Paper Trade J., 114, 43-49 (March 19,1942). (17) Seifriz, W., and Hock, C. W., Ibid., 102, 36-8 (May 7, 1936). (18) Wood, R. W.,“Physical Optics”, New York, Macmillan Co., 1934.
