Some Aspects of Microscopy in Cellulose Research MARY L. ROLLINS Southern Regional Research Laboratory, N e w Orleans, La.
The gross and fine morphology of native cellulose fibers has been studied microscopically ,in a wide variety of physical and chemical environments. Recent technical developments in image formation and in methods of fiber dissection point to the filling of several gaps in existing knowledge. Heretofore, microscopy of the fiber has been limited to staining and swelling methods, together with the use of polarized light. Such examinations, including observations of birefringence, have contributed much to the present understanding of cellulose morphology, but the picture is still incomplete. Phase contrast microscopy, which can eliminate the necessity for staining, has not yet been fully explored, and infrared and ultraviolet microscopy have scarcely been applied at all to cellulose studies. Such methods, aimed at overcoming lack of contrast and of resolution, the main shortcomings of optical microscopy, deserve careful evaluation. The necessity of preparing suitably thin specimens for electron microscopy has led to useful advances in manipulative and microscopical techniques. The development of methods for isolation of individual components of the cell wall for purposes of electron microscopy has permitted interesting observations with the light niicroscope. Quantitative measurements of shrinkage in the isolated primary wall of the cotton fiber confirm its restrictive influence on fiber swelling. The location and orientation of the cellulosic “winding Iayer” is thought to account for its nonreactivity in certain subst i t ution reactions.
cellulose fibers, and such advances in instrumentation and manipulative techniques as are tied in with the essential contribution of light microscopy as the handmaiden of the electron microscope. POLARIZED LIGHT
Light microscopy of vegetable fibers has been limited for the most part to studies of microtome sections and to conventional staining and swelling techniques. With the polarizing microscope additional information may be gained from optical properties which are entirely independent of any morphological features but are indicative of internal molecuIar structure. Refractive index, degree of birefringence, and angle of crystallite orientation are all fiber characteristics which require polarized light for observation. Cellulose crystallites are doubly refractive; this property leads to the production of color effects in cellulose specimens when observed in polarized light, depending on the retardation and interference of light by the crystallites within the specimen. By use of selective retardation in the form of a selenite plate in the optical path, these colors may be changed and intensified to give increased contrast in the color effects(40). Refractive index of a textile fiber is determined microscopicall) in polarized light by the Becke immersion method. The difference between the refractive index along the fiber axis and that across it is known as birefringence; in natural cellulose fibers birefringence is positive, indicating that the greater refractive index lies in the direction of the fiber axis and the lesser index in the lateral direction. According to Hermans ( 2 4 ) the optical properties of cellulose fibers are due to superposition of the optical effects of the elementary structural glucose units in tke cellulose molecular chains. When the longitudinal axes of all chain molecules or crystalline regions in the fiber are parallel to the fiber axis, the fiber would have maximum orientation and xould exhibit maximum birefringence in polarized light. Under this concept, the state of oiientation in a fiber can be described by comparing its measured birefiingence with that of an ideal fiber of perfect orientation and of the same density. There are some native cellulose fi1x.i whose orientation is almost ideal; typical examples are flax and ramie, in which the cellulose molecules are arranged almost cntirelj parallel to the fiber axis. For practical purposes, therefole, it is sufficient to compare the refractive indices of an unknown fiber with those of one of these highly oriented fibers to determine an approvimate orientation figure. The results map be expressed as a ratio of the measured birefringence of the u n k n o m to that of the chosen standard, but it is more instructive to convert the results into a figure for the average angle of inclination to the fiber axis, by substituting the measured refractive index values in the formula for an ellipse ( 3 9 ) . The angle of orientation for cotton, in which certain structural elements are known to have a spiral arrangement, has been found to be approximately 30 degrees to the fiber axis. These measurements which serve to indicate the arrangement of the longitudinal axes of monomeric residues offer means for comparing the orientation of any location along an individual fiber and of different fibers in a sample. Birefringence measurements can be conveniently used to follow changes of orientation which accompany chemical or mechanical treatments of cellulose fibers. The significance of such observations in research is illustrated by the fact that both major and minor refractive indices of mercerized fibers are lower than those of the corresponding indices of the unmercerized control; the birefringence of
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HE ordinary microscope, despite its well-known limitations attributable to the finite wave length of light, has been responsible for providing most of the present knowledge of fiber morphology. Although useful magnifications are limited to about 1500 and the size of the smallest particle that can adequately be resolved is about 0.2 micron, the microscope has been effectively used for the study of both gross and fine features of fiber organization in its natural state and after a wide variety of physical and chemical treatments. Sewer instruments now available are capable of revealing much more about fiber structure, but the light microscope is indispensable in making these newer techniques useful. In spite of the great magnifications possible with the electron microscope, scientists are confronted with almost insurmountable difficulties because of the absorption of electrons by cellulose. Only with extremely thin sections is it possible to obtain images of fine detail and the procurement and handling of such thin fragments of whole fibers tax the ingenuity of the laboratory technician while the interpretation of structural relationships in torn specimens is inevitably open to question. The problem is to reduce the natural cellulose fiber to a condition suitable to meet the limitations of the electron microscope without destroying the natural architectural pattern; in attempts to solve this problem the light microscope is beginning to be used as an accessory tool to aid in dissection o f investigating the microchemistry of Tau‘ materials. This rebirth of light microscopy in cellulose research is the subject of the present discussion, which includes various adaptations such as polarized light and phase microscopy, the possibilities of infrared and ultraviolet light in microscopical investigations of
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V O L U M E 2 6 , NO. 4, A P R I L 1 9 5 4 regenerated cellulose fibers is nearer that of mercerized cellulose than to that of native cellulose. In regenerated cellulose fibers birefringence increases as the fiber becomes progressively better oriented during extension (58)in both drawing and testing, SO that microscopical measurements of fiber birefringence furnish clues as to the extensibility and, correspondingly, the strength of the fiber. In laboratory practice, birefringence is also used as a clue to thickness of specimen. In samples which are known to be of the same substance and to have the same orientation, brilliance of birefringence is taken as indicative of thickness, thinner specimens being less bright. Conversely, in specimens of the same known thickness, brilliance of birefringence is interpreted as indirative of de ree of orientation.
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U LTR 4 1 IO LET MICROSCOPY
In ultraviolet niicroscopy the shorter wave lengths of the incident radiation allox approximately twice the resolving power that can be obtained nith visible light but the equipmwt is relatively expensive, for since glass is opaque to ultraviolet nave lengths the optics of both microscope and illuminating unit must be of high grade quartz, as must also the microscope slides and coverslip$. Sources of radiation are high pressure mercury arc lamps or cadmium arcs. Only a few of the solutions used for mounting media are transparent to ultraviolet light, so that choice of mountant is restricted to sandalwood oil or to specially purified synthetic oils. The poor transmission of glass objectives has been partially overcome by achromatic objectives in which quartz and fluorite have been combined and corrected for spherical and chromatic aberration over the region 2650 t o 2750 A. ( 2 1 ) . A further step in making ultraviolet light feasible as a microscopic research tool is the reflecting microscope, in which the usual difficulties of refracting lenses are obviated by aluminum reflecting surface.; in a two-mirror objective (11, 14, 1.5). A recent application of this reflecting-type objective is the color translating ultraviolet niicroscope originally developed by Brumberg (13) and now being perfected by Land and others a t the Polaroid Corp. (29). At present the technique consists of taking photographs a t three different wave lengths in the ultraviolet. The negatives are then reproduced on color film through three visual color filters. Though the colors of the resulting image are purely arbitrary, they serve to demonstrate, conveniently and vividly, differences n ultraviolet transmission of different parts of the specimen. The fact that various biochemicals demonstrate marked selcbctive absorption a t various wave lengths throughout the ultraviolet has been used very successfully in the examination of fresh biological material (17, SO), particularly in the study of subtle changes in the balance of certain biochemicals within the living cell linked \\ ith specific conditions of disease. In the field of cellulose, however, the ultraviolet microscope still has not been used either as a microscopic or a spectroscopic tool. The absorption curve for cellulose is practically constant throughout, both the visible and the near ultraviolet regions of the spectrum to the far ultraviolet (19) turning upward sharply at 2700 -4. until a t about 2200 -4.absorption of the radiation by cellulose is roughly five times what it was a t 2700 A. I t might be possible to get useful differential absorption in the wave lengths below 2500 A. but the difficulty is to find sources of radiation which produce those shorter wave lengths in sufficient intensity and microscopic equipment which can utilize this type of illumination. Some information could doubtless be obtained with ultraviolet light by use of fluorescent dyes on the fiber in much the same way that biological stains are used in visible light, but it is hardly likely that selective absorption of the cellulose for these substances would offer any new advantages worth the trouble and expense of using ultraviolet light. S o investigations have been
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reported of the impregnation of the cellulose fiber with barium salts or other materials opaque to shorter wave lengths. IYFRARED MICROSCOPY
Microscopical investigations with infrared light have been made possible by the adaptation of the electronic converter tube used in World War I1 Snooperscope and Sniperscope devices ( 5 , 32). Some natural reddish materials and red-stained specimens reveal considerable detail with infrared microscopy (48). Thicker sections may be used than with visual rays and the implication is that such a device would be useful in the study of heavily discolored sections of lignified woody tissues or of very heavily dyed dark-colored textile fibers. Too little infrared microscopy has been done to permit critical evaluation, but cellulose exhibits such high transmission in that region of the spectrum (with the first group of absorption bands appearing a t about 30,000 -4,18)that the infrared technique offers little advantage except through differential absorption of such infrared-opaque dyes as Naphthol green. The use of reflecting microscope objectives in infrared work has permitted some microscopic spectroscopy, but this has not been applied to cellulose. OTHER SPECIAL TECHYIQUES
Interference methods can also be used in microscopy for the measurement of surface irregularities and inhomogeneities in transparent material., but according to hlerton (33) the method is limited to specimens a few microns thick. No specific measurements on cellulose have been reported. Spectrographic attachments are available for the microscope and both absorption spectra and fluorescence may be determined with such devices. Their application to cellulosic materials probably will depend on selective absorption of some ultravioletsensitive substances by the cellulose; no specific work of this sort has been reported. P H 4 S E \IICROSCOPY
None of the special adaptations of microscopy mentioned so far have been exploited in the study of cellulose and few have ever been adequately explored. The phase microscope, on the other hand, seems to have peculiar adaptation to the study of specimens too transparent to be seen in bright field microscopy. A diffraction plate or coating added within the objective and an annular diaphragm below the condenser of the bright field microscope have the purpose of converting slight and invisible alterations of light passing through the specimen into visible differences in image intensity which may be seen and photographed ( 1 2 ) . As pointed out by Barer (IO), the use of polarized light with the phase microscope increases the contrast obtainable in many cases. I n the field of cellulose the phase microscope has found its greatest usefulness in the examination of rayon fibers (44). In cross sections of viscose rayon the skin effect can be seen readily without any staining and the distribution of solid pigment particles or of gas bubbles or foreign inclusions in the body of the fiber can be studied and recorded easily. In the case of cotton it has been found that isolated cell Kall fragments can be examined without any mounting medium, thus making it possible to observe specimens in the same desiccated condition in which they are photographed in the electron microscope, for better correlation between observations made with the two different instruments. EXPERIMENTAL RESULTS WITH COTTON FIBERS
Microscopical approaches have been variously applied in fundamental investigations of cellulose fibers for approximately a century. The extensive work on the cotton fiber in the first quarter of the 20th Century by Balls (6, 7 ) served to stimulate
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cotton fiber morphology as summarized by Flinl (SO) wa9 supported by considerahle evidence from m m v indenendent investieators. Trino’r (47) schematic diagram of fiber architecture represents the cell wall as a lamellate struetux composed of outer membrane or primary wall winding layer, seoondary wall, and lumen 01 central canal. From electron microscope studies the primarv wall and cuticle have been estimate0 t o be about 0.1 micron in thickness and the winding layeyer of the secondary wall not morf than about 0.2 micron. The main body of the secondary wall is probably between 4 and i microns in thickness and is composed of many layers whose thickness varies with many factors of growth. Each layer is believed to be made up of microfibrils of cellulose hranching and rehranohing, but roughly parallel and arranged in a more or less spiral fashion about the main axis of the fiber. In the first layer of secondary thickening (the winding layer) the spirals are much ooarser and spiral in a direction opposite to those of all t h e various layers of the main body of the secondary wall. At the center is the callapscd tubular central canal containing the remains of cell protoplasm. I
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Figure 1. Cotton Fiber Stained with Nile Blue Sulfate and Swelled with C\ipriethylene~iamineHydroxide
I n general, this picture of the layered morphology of the cotton fiber cell wall is confirmed for various other types of plant cell walls by the observations of Van Iterson (49),Aldaba (I), Bailey (8, 9), Anderson (S), Ritter (42),Rollin8 (48), and others. None of these features is visible in raw, unswollen fibers, even under high magnifications. Swelling in such agents as 70% sulfuric acid or the cupritetrammine bases is necessary to reveal structural details in native fibers; the pattern which can he observed in the swelling of chemically modified cottons in appropriate solvents is very similar. Because of its pectic content the primary wall may he stained with an aqueous solution af ruthenium red, methylene blue, or Nile blue sulfate, the basic dyestuffs reacting with the carboxyl groups present; they d80 stain the proteinaceous lumen contents for the same reason ($6). The wax complexes of the cuticle can he colored with such fat stains as Sudan black and oil red but satisfactory intensity is achieved only after the fibers have been treated briefly with boiling ethyl alcohol (35,47). By such conventional swelling and staining techniques the gross morphology of the fiber can he demonstrated in longitudinal view, as in Figure 1, which shows a mature cotton fiher, swelled with cupriethylenediamine hydroxide to show the various fiber components. The primary wall, stained with Nile blue sulfate, can he seen disappearing a t the outside edge of the fiber in the form of ruptured fragments, and the winding layer with flecks of primary wall clinging to it, wraps around the fiber. Within the winding layer may he =en some of the
ions of Cotton Fihers A.
Single fiber swollen to show growth layer. B . Bundle of mature fibers C. Bundle of imrnaiur~fibers
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iayers of tho secondary wall, and a t the center the dark-stained lumen and protoplasmic residue. In the greatly swollen cross section a t the left in Figure 2 the growth layers can he plainly seen, while a t the right are illustrated collapsed cross sections of dried fibers. The fullbodied mature fibers, a t the center, whioh received their full complement of cellulose during growth, are contrasted with the collapsed sections of the undernourished, immature fibers a t the right. One of the simplest laboratory tests far t,his cell wall maturity is the ASTM method (S) using polmized light, in which the criterion of cell wall thickness is the amount of retardation of the light upon passing through the fiber. This test is illustrated in Figure 3 in which the lower fiber i8 mature and thiclc-walled, the upper, thin-walled and immature. When placed in the proper position in the field of a microscope equipped with crossed PJicols and a first-order red selenite plate, the brilliantly birefringent thick-walled fiber transmits a green or yellow color and the thin-walled fiber a purple or deep blue color. The picture also illustrates an interesting phenomenon of crystallite orientation in cotton fibers. Although the microfibrils in cotton lie roughly parallel to the main axis of the fiher, they spiral around the axis in a helical manner. Thc direction of the helix reverses many times along the length of the fiber and when the fiber is observed under the microscope in polarized light these points of fibril reversal appear as dark extinctiun hands in the hrilliantlJbirefringent fiber. Wakeham ( 5 0 ) has concluded from statistical considerations of the frequency of rupture a t reversal points during tensile breaks that these are probably meas where th
