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SPIERER LENS and
COLLOIDAL STRUCTURE FIGURE 1. ILLUSTRATION OF THE PRINCIPLE OF THE SPIERER LENS FIGURE 2. ELLIPSE OF LIGHT DIFFRACTEDBY A COLLOIDAL PARTICLE
WILLIAM SEIFRIZ University of Pennsylvania, Philadelphia, Pa.
The greatest intensity is in the direction of the illuminating ray (from below).
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Pictures of colloidal matter produced by the Spierer dark-field lens represent a true structure when suitable material is used and the optical system is properly handled. The major types of structure observed have been seen by several other methods. However we regard the Spierer picture, it can exist only in virtue of a definite and corresponding type of structure within the material observed. The striated structure seen in cellulose represents a fine and naturally occurring grating. The Spierer lens reveals several distinct types of structure in colloidal matter. Proper handling of the lens produces a sharp picture, devoid of diffraction lines, of so small an object as a coli bacillus. Improper handling, or unsuitable material, produces artifacts. Recent x-ray work on cellulose gives evidence of a structural unit comparable in size to the supermicelles shown by the Spierer lens.
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FIGURE 3. DIAGRAMS OF SPIERER, A, AND CARDIOID, B, SYSTEMS, ILLUSTRATING WHYTHE SPIERERLENSPICKS UP MORE OF THE MOSTINTENSELY SCATTERED LIGHT(FIGURE 2) THAN DOESTHE CARDIOID SYSTEM;C Is LOSTIN THE LATTER
HE purpose of the present article is to clarify some of the misconceptions which have arisen in regard to the Spierer lens, to elucidate further the optical principles on which the lens rests, and to show that the types of structures revealed by the lens in diverse forms of colloidal matter are real and capable of verification by other methods. The Spierer lens has been described by its inventor (9)and by others (7). Briefly, it is a dark-field optical system consisting of an oil-immersion objective into which a metallic mirror has been inserted. The mirror is centrally located on the front lens and covers about one-seventh of the total aperture of the objective. The illumination is a small pencil of light coming from below; all of the direct light thus received is totally reflected by the mirror. The scattered light is picked up by the periphery of the lens (Figure 1). The advantage of this type of dark-field illumination has been explained before (7, 9 ) , and a mathematical interpretation (based on Rayleigh’s formula) is given by Thomas (1Q.l Numerous kinds of matter have been studied with the aid of
the Spierer lens; among them cellulose has received most attention. One of the misunderstandings which has arisen in regard to the Spierer lens has to do with the principle on which the lens rests. Clifford and Cameron (3) raise the question: Why, if it is the reflected light from the mirror in the objective which is effective, any type of vertical illumination, such as in the metallurgical microscope, will not do as well? This would be true if the reflected light from the objective (i. e., from above) were the effective beam, but it is not. AS has been pointed out (7), Spierer designed the lens on the assump1 Whether or not the Rayleigh formula, based on ”infinitely” small particles, is applicable here, cannot be definitely said. Just how amall ”infinitely small” is would have t o be determined. The formula may hold, but less accurately so, for large particIes.
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4. TYPICAL STRUCTURE OF SMALL PART Pleurosigma TAKEN WITH SPTERER LENS
FIGURE
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A . Light field;
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FIGURE 5. PART OF SWFACE OF CELLULOBE WALL OP A LIVING PLANTAUaurn) CELL TAKEN WITH SPIEnmR LENS, SHOWINGERFECT PARALLEL ORIENTATION OF STRIAE, EXCEPT AT EXTREME LEFT W E ~ E EORTENTATIDN IS DISTURBED BY STRAIN
f?'
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R . Spierer dark field
sults. Clifford and Cameron raise the further question as to why, if i t is the beam of light coming from below that is effective, the cardioid condenser should not be better. They assnine that in the Spierer optical system "the scattered light in the direction of its maximum intensity cannot enter the objective on account of the mirror," while in the cardioid the light rays impinge on the object obliquely; "hence, the light in the region of its maximum intensity (in the direction of the illuminating ray) enters the objective." Thus the cardioid should be better if the principle on which the Spierer lens is based is .sound. Figure 3 explains this point better than words.
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WITH. my new apparatus ohservations should be niude on material whoso properties are h o r n . Spierer showed that, the well-known structure of the diatom, Pleurosigma, is reproduced by the Spierer lens precisely in its familiar form (Figure 4) with no duplication of lines ("pearl rows"). The Spierer lens bring8 out the structure more sharply and over a greater area than does the usual liglitX~eld oilimmersion objective. Some other types of test objects, such 8s butterfly wings and fine glass rods, are not reproduced by the Spierer lens without artifacts, but this is true of a.11types of dark-field illumination (and of light-field illumination with an oil-immersion lens as well). Dark-field methods in general are possible only because of the diffraction of light; consequently, objects which are troublesome with any type of optical system because of diffract.ion phenomena become much more so with a dark field. ( B u t t d y wings m e e s p c i d y nnftworable material for any kind of dark-field study because of the air-6lled spaces--tracheal tubes-they contain.) The dark-field method is applicable with the best results to very thin objects
whose principal surfaces lie flat. Properly prepared cellulose membranes correspond with this description. Wowever, observations on fibers (e. g., of cellulose) give dependable results if one is exceedingly cantious to distinguish between colloida1 structure and extraneous diffraction lines. This can be done by comparison with the light-field picture. The Spierer lens contains an iris diaphragm permitting frequent and immediate comparison between the dark- and the lighb field picture. When the Spierer lens is used in combination with an Abbe condenser, all gradations between a dark and a light field can be obtained by opening the iris diaphragm below the stago more or less; in this way the differencesbetween the normal light-field image and all degrees of dark-field illumination can be followed; the correct interpretation of the image is thus made possible. A FURTHER criticism directed against the Spierer lens is that the pictures obtained are not only diffraction phCnoInena but bear no relation to the real structure. First, attention should be called to the fact that diffraction phenomena are reproduced by all types of optical systems for ndcroscopical work. The usual oil-immersion objective has reached its useful limit in resolving power, and, when the lens is pushed beyond this limit, diffraction lines result in light field as well as in dark field. Light-field pictures of macerated cell walls are often a confusion of diffraction lines; these lines are accentuated when viewed in a dark field. Furthermore, throwing a lens out of focus is a classical way to produce artifacts. The minute circular perforations in an Abbe test plate, when viewed witli a light-field immersion lens, first assume the shape of hexagons, then are multiplied in number, and finally ta.ke on the appearance of diagonal lines as the
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lens is raised out of focus. We do not, therefore. discard so ilsefut a tool as the oil-immersion objective. The only possible hasis for the contention that a Spierer picture bears no relation to the object viewed is that the picture is formed by the lens. This criticism is untenable because the Spierer lens produces a picture only when the light received is daracted by intervening colloidal material. Moreover. the nictures obtained are not alike. as shown bv Figures 5,’ 10, abd 11. Granting, then, that the Spierer picture is produced by the material viewed, the next point to he considered is whether or not the picture is a reel one or an artifact of diffraction lines. If we tentatively admit the latter to be true, the obvious question is, diffrsction lines of what? This question has been neglected or evaded by the critics. The first thought is that the l i e s seen (e. g., in Figure 5 ) are duplica, tions of fewer lines. One point is gained herenamely, the admission that to obtain two lines it is neewary to have one to start with. The 6rst picture of cellulose taken with the Spierer lens (ETgure 5) is the surface of the wall of a living plant cell. If it is true that the many lines are duplications of fewer lines, then the Spierer lens revals at least the type of structure present, merely duplicating it, so to speak. But let us see if the real grating is necessarily less fine than that shown by the Spierer lens. First, this wmfound not to he the utse in the Spierer picture of the diatom, Pleurosigma, the number of “pearl IONS” bemg the same in the dark field as in the light field. If we view a coarser and a 6ner grating, we find no duplication in the former and duplication in the latter. A micrometer scale (with spacings 0.01 mm. apart) observed through the Spierer lens and then through a mrresponding one-twelfth oil-immersioo light-field objective shows the same number of lines in both cases. If a much 6ner scale is observed-for example, a diffraction grating of twenty t h o u m d lines per inch-the number of lines shown by the Spierer lens will he seen to be exactly double that with the corresponding lightfield objective (Figure 6). Again we might say that the Spierer lens has shown the type of structure present but has amplified it hy duplication. This may be the case, or the lens may have analyzed the grating-i. e., have caught the light scattered by the edges of the linesand, since each line has two edges, twice as many lines appear in the Spierer picture a8 in the light-field picture. Abbe f f ) in reference to light-field work said: “The smaller the linear dimensions of a structure, the more indefinite are the conclusions concerning the real structure which one can draw from the microscopical image.” While it is true that diffraction lines are frequently produced by the Spierer lens, it is equally true that they can he wholly avoided under favorable conditions -vis., when the
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differences between the optical constants of the object and of the medium are small. Even in the case of as small an object as a coli bacillus (Figure 7),interference stripes can be wholly eliminated. The material in which the coli bacillus is suspended io Figure 7 is a culture of bacteriophage. I t is possible that the brightly illuminated particles are individual bacteriophage, two of which can be seen adhering to the body of the bacillus. T H E fact which has perhaps most disturbed the critics of % the Spierer lens is the difficulty io understanding why any optical system should show similar, a t t i e s identical structure in otherwise diverse material. The first instance of this sort was the apparent similarity in structure of such obviously diverse systems as cellulose and protoplasm. The similarity is, however, purely superficial. When the microscopically visible strnctural units of cellulose are unsymmetrically arranged, as a t the extreme left of Figure 5 or as io parts of Figure 10, the structure closely resembles that of an emulsion. When protoplasm is viewed either with a Spierer lens (Figure 8) or with light-field illumination, it too has a mottled a p p e a r m c e i . e., of au emulsion. The superficial similarity is further emphasized by the appearance in protoplasm of a structure like that of oriented particles in cellulose. When protoplasm is under tension, as when stretched into a thread or when streaming, its emulsion globules become distorted; they then resemble short rods placed end to end in parallel IONS (Figure 9). This structure is similar to Spierer pictures of certain forms of cellulose (Figure lo), but the two systems have nothing in common except a purely superficial resemblance. Other instances of apparent similarity in Spierer pictures are shown in Figures 5 and llC, and in 10 and 11B. The resemblance is pronounced, but this is to be expected since the one material, coal, came from the other, cellulose. Clifford and Cameron object to the similarity in Spierer pictures of paper pulp and asbestos, though both are built up of fine fibers. Their chemical dissimilarity is not of significance. A grating or a brush heap made of cellulose may show a structure identical with that made of any other material, Systems as diverse in chemical constit.ution as gelatin, cellulose, and soap curds, have had identical colloidal (micellar) structures postulated for them (i. e., linear molecules or aggregates). As a matter of fact, Spierer pictures are not identical. The excellent Spierer pictures hy Thiessen (10)show different structures in coals (lignite, anthracite, etc.) and in cellulose; thus, in Figure 11A there is a scattered arrangement of parts; in B the structure is made up of short rods which are, in the main, symmetrically oriented in parallel rows; and in C the structure consists of continuous and parallel fibrils. E best evidenceof Spiererpictures (those T T.Hwhich . artifacts . aretherealityof eliminated or reduced to a miniin
FKGIIRX 10.
PORTION OF T W O ADJOINING CELLa (VERTICAL CROSS WALL I N CENTER) SHOWINQ A LESSWELLrOROANIZEDSTRUCTURE OF CELL-LOSE; SUPERMIC~LLES AnE NOT IN PERFECT
AWGNMEXT
mum and then distinguished from that which is real) is their reproduction by other methods. In the careful and thorough work of Thiessen it W&S not possible to obtain the structures seen with the Spierer lens by any other method of observation. But in other work the Spierer pictures were later found to have been reproduced in almost identical form by two other types of optical systems. In the caseof protoplasm the two phases of the very delicate ernulsion differ so little in refractive index that they were overlooked in the light field. Only later were they discovered iix an admirable photograph by Scarth (6) taken in light field. The photograph of protoplasm taken by Scarth is not reproduced here because the optical differentiation between the two phases of the hyaline protoplasmic emulsion is too slight to he seen in an ordinary
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Further verification of Spierer pictures is to be had in lightfield observations of paper pulp fibers. Fibers in well-beaten paper pulp are, in light field, seeu to be made up of fibrils running parallel with the fiber axis, exactly as shown by Thiessen for the dark field (Fignre 1lC). That the fibrils are delimte fibers and not. surface irregularities or artifacts is evident from the frayed ends of the composite fibers. The existence of fibrils is further demonstrated by the technic of microdissection; a single fiber can be dissected into its component fibrils with the aid of glass microneedles mechanically operated under the high power of the microscope. Langelaan (4), in a recent article on striped muscle, states that there is full agreement between light field and Spierer dark field in the major aspects of the muscle structure, with the Spierer lens bringing out greater detail. CERTAIN critics of the Spierer lens find support for 4 their comments in a publication by Siedentopf (8). He demonstrated that a microscope objective having a central stop gives dark-field images with two defects: The interference lines around the object (e. g., a colloidal granule) are so luminous as to be misleading; and when a very fine grating is examined, double the number of lines which really exist are seen in the grating. These considerations are based on an arbitrary example of a central stop which masks half the aperture of the objective. Siedentopf himself recognized this fact and stated specific& that “them defects hold for the condition where the central screen covers exactly o n e half of the aperture.” In the Spierer lens only one-seventh of the aperture of the lens is mssked by the central mirror This relation reduces the interference defect to a minimum and is nediaible in most instances. The manner in which FIGURE11. SPIERERPHOTOQRAPIB OF COAL -D CELLUWBE(BY R. THIEBBF~N) SHOWING TEREED10TINCT TYPESOF STRUCCURE 1.scatteered, paniclea in COQI: B . 8 u ~ m i c s U e soriented into parsllel striae, m coal: C. Fibrillae arranged in perieot parallelism in ramie flber ( X 1WOl.
reproduction; only the Spierer lens brings out the contrast vividly, as in Figure 8. Confirmation of the Spierer picture of cellulose was found in pictures taken some years ago by A. Herzog of “cellulose silk” and “viscose silk.” Hersog’s photographs (Figure 12) were taken with a Zeiss-Siedentopf cardioid condenser. Comparison of Figures 12A with 5 or 11B and of 12B with 11A show that typical Spierer pictures are obtained by one of the best known dark-field optical systemsnamely, tho Zeiss-Siedentopf cardioid condenser. A convincing bit of evidence in support of the presence of striae in plant cell walls, such as are revealed by the Spierer lens, is that of Astbury and eo-workers (8). They show a photomicrograph and an x-ray pattern of the cell wall of the alga, Valunia. In the photomicrograph two sets of striations are superimposed upon each other, resembling two diffraction gratings; the lines cross a t an angle of 80”; this picture a p pears to be an exact replica of the Spierer picture in Figme 5, both of natural cellulose but seen through different optical systems. Astbury’s x-ray photograph of the same Vuloniu wall shows that the striae are parallel to the direction of the cellulose chains. Astbury concludes that the fiber orientations nerskt through the whole thickness of the cell wall (i. e., are not superficial). Similar illustrations in a recent article by Bailey and Kerr (SA) are photographs taken with polarized light; they show alternating brilliant and dark layers in the cellulose wall of nlant cells. Bailey and Kerr’s Fiaure 13. showina alternating I
FIGURE 12. PROTOGRAPES or “CELLULOSE SILK” ( A ) AND “VISCOSESILK’’( B ) TIKENWITB ZEISSSIEDENTOPF CAEDIOID (DARK-FIELD) CONDENSER (Photographs me by A . Hercog. from Oetwald’a book (a), pubiiahed wit4 the iatter’a kind permission.)
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A B C D ILLUSTRATING PRINCIPLE OF SPIERER LENSAS COMPARED WITH SIEDENTOPF CENTRAL SPOTLENS FIGURE13. DIAGRAMS
image of the object. The central maximum, I, also contributes to the formation of the image, which makes it more complete. Figure 13B shows (in comparison to A ) a central spot lens where the dimensions of the spot correspond to those which Siedentopf took as a basis for his calculations (the central spot, which is opaque, masks about one-half of the aperture of the lens). All other conditions being equal, the central maximum is held up entirely by the black spot which is fully opaque. The first lateral spectrum is also held up, and only the second lateral spectrum (3, Figure 13B) passes through the lens. These are bad conditions for the formation of the image. Figure 13C shows how the same object, illuminated the same way, can again give an incomplete or false image, in spite of the fact that the central spot is small. This time the lens is weaker (its focus is longer), and one sees imlqediately that under such conditions the second lateral spectrum (3, Figure 13C) passes outside the lens, in spite of the fact that the angles between the spectra remain unchanged. In Figure 130 the lens is the same as in C, and the central stop as large as in B3 but this time the object is changed (the grating is coarser and the space between two lines greater). In the case of coarser gratings the diffraction spectra are closer t o each other and the angles between them are smaller. Consequently both lateral spectra can enter the lens and pass through it, and we obtain a true image of the coarser grating. Siedentopf was not only aware that his calculations hold only when the central spot covers “exactly one-half of the opening,” but also that the optical reasons for this phenomenon aye as just given: “It is well known from Abbe’s theory on the formation of microscopical images that the distance between the lines of a grating is correctly reproduced only when at least two successive spectra, out of the total diffracted light, become effective in the formation of the image.” These difficulties are, in the main, avoided by the Spierer lens for the Seasons noted and illustrated in Figure 13A. WITHOUT entering into a discussion of the controverproblems involved in cellulose chemistry and theories of colloidal structure in general, it is nevertheless necessary to refer briefly to the matter of micelles in order to straighten out the confusion which Clifford and Cameron have produced in regard to the writer’s use of the term “supermicelle.” Realizing that the microscopic units seen in a Spierer picture (the short rods which, oriented end to end, build up the articulate fibers in certain types of material, as in Figures 5 and 11B)are not the micelles the chemist has in mind, the writer coined the term ‘LsupermiceUe.” Clifford and Cameron (3) state : “These rod-shaped particles are,
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according to Seifriz, supermicelles which are probably aggregations of the crystallites postulated from x-ray data.” Yet in the next paragraph they add: “It appears that the spacings are comparable to the dimensions of the micelles shown to be present in cellulose by x-ray observations.” Obviously these two statements are contradictory, yet Clifford and Cameron make the second one the basis of their criticism when they state that their investigation “does not support this interpretation.” They might have added, “nor do the investigations of Seifriz.” If the use of terms is to be the basis of criticism, let us see if too critical a n attitude toward the micelle is justified. The micelle as a unit of colloidal structure has seen many vicissitudes. Naegeli, who coined the word, made no definite reference to its size. I n cellulose the micelle must be at least as long as the cellulose molecule, for it is by definition a bundle of the latter. The cellulose molecule, and therefore the micelle, has grown from the minimum of 400 A. given by Meyer and Ma&, to 2000 A. suggested by Staudinger, and now 1.7 p (17,000 A,, with a molecular weight of 500,000) given by Kraemer and his co-workers (based on ultracentrifugal and viscosity; investigations of molecular weights of cellulose in cupramrnonium). The last value of 1.7 p is very close to the length given by the writer for the Spierer particles or supermicelles in natural cellulose-namely, 2 p (the same length was given by Thiessen). This value is within microscopic visibility.
Literature Cited (1) Abbe, E.,Gesammelte Abhandlungen, 1, 84 (1904). (2) Astbury, W.T., Marwick, T. C., and Bernal, J. D., Proc. R o y . SOC.(London), B109, 443 (1931). (2A) Bailey, I. W.,and Kerr, T., J . Arnold Arboretum, 16, 273, Figs. 5 and 12 (1935). (3) Clifford, A. T., and Cameron, F. K., IND. ENG.CHEM.,26, 1209
(1934). Arch. nderl. physiol., 19,445 (1934). (4) Langelaan, J. W., (5) Ostwald, Wo., “Licht und Farbe in Kolloiden,” Dresden, 1914. (6) Scarth, G.W., Protoplasma, 2, 189 (1927). (7) Seifriz, W.,J . Phys. Chem., 35, 118 (1931). (8) Siedentopf, H.,2. wiss. Mikroskop., 26, 391 (1909). (9) Spierer, C.,Arch. sci. phys. nat., 8,21 (1926). (10) Thiessen, R.,IND.ENG.CHEM.,24, 1032 (1932). (11) Thomas, A. W.,“Colloid Chemistry,” New York, 1934. RECEIVED June 1, 1935.
