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Transmission electron microscope studies and their relation to polarizing optical microscopy in rat tail tendon
1978 Vol.: 9, pp.71-82. © Pergamon Press Ltd. Printed in Great Br ta n
0047-7206/78/0601-0071 $02.00/0
Micron
TRANSMISSION ELECTRON MICROSCOPE STUDIES AND THEIR RELATION TO POLARIZING OPTICAL MICROSCOPY IN RAT TAIL TENDON J. DLUGOSZ, L. J. GATHERCOLE AND A. KELLER H. H. Wills Physics Laboratory, University of Bristol, Tyndall Avenue, Bristol BS8 1TL, U.K.
(Received 17 November 1977; received for publication 20 January 1978)
Abstract--An investigation on rat tail tendon is described which follows the periodic banding pattern seen by the polarizing light microscope down to the level of transmission electron microscopy. This work was carried out by longitudinal sectioning of preselected tendon portions in preselected orientations around the tendon axis. In the course of this, full bridging of light with electronmicroscopic structural information has been achieved. In particular, complete verification is provided for the planar crimp structure as originally postulated from polarizing microscopy. "l?hus thin sections fully conformed even to the idealized crimp representation when viewed under the polarizing microscope, while the crimp sites themselves could be detected. These were seen to be sharp as implied by the rigid hinge model, which was invoked previously merely as a mathematical convenience to account for the mechanical behaviour. This sharpness of the crimp site persisted down to the level of the 68rim banding visible under the electron microscope and was seen to occur within 1-2 such band periods. Various individual features seen at all levels are discussed.
INTRODUCTION The main features of crimping in collagenous fibres of extensible connective tissues have been elucidated by ourselves and co-workers (Diamant et al., 1972; Gathercole and Keller, 1975; Baer et al., 1975; Torp et al., 1975). The aim of the present paper is to reveal the crimp sites by transmission electron microscopy (TEM) in order to confirm their existence directly, to obtain information on their nature and thus open up the possibility of studies including possibly electron diffraction (Gathercole et al., 1978a). If realized, this would complet ethe continuity of information gained from polarizing microscopy (Diamant et al., 1972) X-ray diffraction (Gathercole and Keller, 1975; 1978a)and scanning electron microscopy (Gathercole et al., 1974) from the fibre to the fibril level in rat tail tendon (RTT). As will be apparent, this has been achieved.
without teasing and cut into 5mm lengths. These were fixed in standard cacodylate-buffered 12% aqueous glutaraldehyde pH 7 at room temperature for 20min. Dehydration was in successive changes of 33%, 66% and 100% ethanol with final permeation in Spurr (1969) resin monomer. Samples were then permeated with Spurr reactive mixture and polymerized. Embedded samples were examined by polarizing microscopy to check for the retention of crimp and to determine the orientation of the crimp plane locally in the areas it was intended to cut. Sections were trimmed and then cut on an LKB Ultramicrotome, with continuous visual monitoring and adjustment to ensure cutting was parallel to the crimp plane. Sections were cut a few at a time, then examined under crossed polars to check the crimp plane alignment. Some sections were also cut at random angles to the local crimp plane. Sections were cut with knife movement both parallel and perpendicular to the bulk fibre axis in order to investigate the influence of cutting direction on the shape of the crimp site.
EXPERIMENTAL Tendon units of 6-month old rats were released from surrounding tissue in the tail 71
72
J. Dlugosz, L. J. Gathercole and A. Keller
TEM and Polarizing Optics of Rat Tail Tendon Sections were stained with 1 ~ aqueous uranyl acetate, and examined in a Philips 301 electron microscope. Some sectioning was carried out also with the section plane perpendicular to the fibre axis but because the corresponding information, plus more, was also contained in some random longitudinal sections (namely where crimp plane is perpendicular to the section plane) this will not be illustrated separately. RESULTS AND DISCUSSION Optical microscopy Figure 1 shows the block from which sections were cut containing a unit fibre of RTT with crimp plane lying in the section plane. This corresponds to the p -- 0 ° position of Gathercole and Keller (1978a) viewed in incident light. In this case, illumination is from above along the fibre axis at an angle of about 40 ° to the fibre. The appearance is surprisingly similar to that obtained with transmitted polarizing optics with one arm of the crimp parallel to polarizer or analyser (see for instance Diamant et al., 1972). These are transverse bright and dark bands due to specular reflections from surfaces composed of bundles of parallel fibres sharply bending into and out of reflecting position. These bands lie within and throughout the fibre, and their contrast can be made to reverse by rotating the illumination direction through 180 ° with respect to the fibre. The optical behaviour of this tendon shows a close resemblance to that displayed by the semiprecious gemstone 'tiger's eye' most strikingly displayed, when, as we have done, the two are viewed side-by-side. It is known that, in this mineral, sheets of parallel fibres bend sharply in varying directions, giving rise to lustrous cross bandings of brown-gold which move with changes in viewing direction (chatoyancy). This too is a fibre plus matrix system, the kinked fibres of crocidolite being embedded in a matrix of silica, thus providing a documented example of the phenomenon we are observing in our tendons.
73
Figure 2 shows a specimen block viewed in combined transmitted and incident ordinary light, from which about half the fibre has been removed by stripping. Here the crimp sites are seen directly. The sites show visually that there exists a continuous gradation in sharpness of such sites. Similarly, sites originating as very sharp on, say, the extreme left hand side of the fibre at A may become smoother as they traverse the fibre to the opposite edge B. In this, the pictures are in agreement with an earlier SEM picture invoked in support of the interpretation of complex features in the low angle X-ray patterns related to the 68nm periodicity (Gathercole and Keller, 1978a). A similar specimen is seen in Fig. 3 viewed in transmitted ordinary light. Here crimp sites are seen directly, though there is an indication of complexity in that there is evidence of an underlying reversal in the direction of the fibrillar texture at crimp sites. Such reversals are indicated by arrows (see also Fig. 4). A similar specimen is seen in Fig. 4, viewed in transmitted light with crossed polars. Figure 4a shows the specimen with the bulk fibre axis parallel to the polarizer or analyser while Figs. 4b and 4c show the stage rotated so that alternate crimp arms are parallel to the polarizer or analyser. This shows some differences from the behaviour expected from the simple model of Diamant et al. (1972). In the first instance, broad dark bands are not seen on rotation of the stage by 0o; instead, alternate crimp sites, originally dark, become bright, though their position is still obvious. Similarly, there appears to be a more deeply situated fibrillar texture running some half crimp wavelength out of phase with that obvious on the surface. Such features have first been noted in glutaraldehyde fixed adult specimens soaked in hexylene glycol (Kastelic et al., 1978) and subsequently also in fresh, wet 3-week old specimens (Gathercole and Keller, 1978b). It is interesting to note, however, that in Fig. 4, the crimp sites are fully traceable to the edge of the tendon unit and that at the edges there is no uniform appearance of flattening
Legend for Paoe 72 Fig. 1. An embedded RTT unit fibre viewed in incident light showing marked chatoyancy of the crimps. Direction of illumination is down the fibre along the axis at ,~40 ° into the fibre, x 100. Fig. 2. An embedded RTT unit fibre from which about half has been stripped. Viewed with combined transmitted and incident light x 100. Fig. 3. Enlarged view of a fully embedded RTT in transmitted light showing crimp sites. The crimp is out of phase in the lower half of the tendon; this is seen diffuselyand is indicated by the marker arrows on the left. x 370.
74
.I. Dlugosz, L. J. Gathercole and A. Keller
J
TEM and Polarizing Optics of Rat Tail Tendon out of the crimp--an observation of some relevance to current considerations (Kastelic et al. 1978) of how the crimped unit may constitute the tendon (a point not to be pursued here). All this is in marked contrast to the appearance of sections taken from these blocks when such sections are viewed in the polarizing microscope. Sections for this purpose were cut to a thickness of about llam on the Ultramicrotome. Figure 5 shows the appearance of longitudinal sections with cutting direction perpendicular to the fibre axis. Figure 5a shows the appearance with one arm of the crimp parallel to the polarizer or analyser, Fig. 5b the median position, with only the very narrow crimp sites themselves in the extinguishing position, while Fig. 5c shows the alternate arms of the crimp parallel to the polarizer or analyser direction. As can be seen, the simple behaviour of Diamant et al. (1972) is followed. The bands change from narrow to broad on stage rotation, the crimp sites are very sharp and the planarity of the crimp is self evident. Clearly, whatever the packing on a larger scale it is possible from these pictures to obtain coherent sheets of planar crimping fibrils that extend over the whole width of the unit fibre. Sections were also cut parallel to the main fibre axis. The general appearance was the same as the specimens cut perpendicular to the axis, except that the crimp is further compressed. In practice, it was found to be easier to obtain coherent longitudinal sections with the knife edge moving perpendicular to the fibre axis. Electron microscopy The sites displayed as in Fig. 5 can be viewed directly in the electron microscope. Figure 6 shows a low magnification view of an ultrathin section cut from the same block as in Fig. 5. There is a sharp kink within each adjacent fibril and alignment between adjacent kinks. The cutting direction is as shown, approximately parallel to the lower arm of the crimp. Figure 8 shows a similar area where the crimp has an overall smooth appearance. Here cutting direction very nearly bisects the angle enclosed by the two crimp arms and this may well have contributed to a 'flattening' of crimp with a corresponding
75
smoothing out of the discontinuity at the crimp site. The crimps in individual fibrils are still sharp, though these deflect through a smaller angle and are less well aligned, contributing to an average smoothness. Higher enlargement micrographs (Figs. 7 and 9 respectively) readily reveal the striations corresponding to the 68nm periodicity familiar from the fine structural electron microscopy of collagen. The main points to note are as follows. First, the existence of the crimped structure has now been demonstrated by transmission electron microscopy. Second, that the crimped arrangement is definitely planar otherwise the individual fibres could not be seen along any appreciable length within a given section (in fact they are seen to extend over the full section) and third, that the crimp is indeed sharp. All these features were previously deduced from polarizing optics of the full tendon units (Diamant et al., 1972; Gathercole and Keller, 1975) and/or inferred (the sharp hinge of the crimp) from the mechanical model constructed to account for its mechanical properties. Fourth, the dimensional level has been reached where the 68nm period has become visible simultaneously with the crimp site. We see therefore that the present work successfully extends the previous low power microscopic observations and the inferences from the mechanical behaviour to the level of the electron microscope. This establishes complete continuity between the macroscopic, microscopic and ultrafine structure research methods transferring the results of the former to the latter level. Examination of the 68nm bandingyields further information. The bands are skewed with respect to the fibril axis and this is in different directions in the corresponding crimp arms for parallel and perpendicular cutting (Figs. 7, 9). Also for a given cutting direction this skewing is of opposed direction in the two crimp arms (all these effects are shown schematically in Fig. 10). All this indicates that the skewing was largely induced by the cutting, although the existence of much smaller amounts of skewing along the crimp arm parallel to the cutting direction (Fig. 7) suggests that some obliquity may have also been present
Leyend for Paye 74 Fig. 4. Same specimen as in Fig. 3 viewed in transmitted light with crossed polars, (a) with the bulk fibre axis parallel to analyser or polarizer, (b) and (c) with alternate crimp arms parallel to polarizer or analyser. All x 190.
76
J. Dlugosz~ L. J. Gathercole and A. Keller
T E M and Polarizing Optics of Rat Tail Tendon CRIMP SITE
C
FIBRIL
(Q)
~
CRIMP SITE
FIBRIL.
Ib) CUT
Fig. 10. Diagram showing the origin of shear effects in crimp compression and 68nm band tilting as shown in Figs. 6 and 7 (10a) and 8 and 9 (10b). The central region shows a single fibril within a suggested array of parallel fibrils (not to scale).
ab initio in the native tendon (the possibility of this latter effect has been raised in connection with our earlier X-ray work (Gathercole and Keller, 1978a)). Concentrating on the more pronounced effects which are definitely induced by the cutting process, we note that the shear is such that the 68nm bands tend to align with the edge of the knife. Thus in Fig. 7 they become almost parallel to the line containing the crimp site while in Fig. 9 they tilt in the opposite direction. This is shown simplified in Fig. 10. We can consider this as resulting from shear deformation on compression, the direction of which is along the movement of the knife. It will be seen that if planes of easy slip exist along the fibril direction then such slip, as activated by the compression, would produce the observed direction of skewing of the bands in all cases. Slippage along the direction of the tropocoUagen molecule is of course reasonable, although the readiness by
77
which this takes place may be noteworthy. It is important, however, to note that the preservation of the banding on such sqppage implies that the units which slide past each other already possess the 68nm periodicity. Thus it cannot be on the scale of the triple helix itself but it must involve an appropriate multiple of it such as already embodies the quarter stagger believed to be responsible for the 68nm banding. A point to the same effect has already been made previously (Gathercole and Keller, 1978a). Observations can also be made concerning the register and numbering of 68nm bands across crimp sites. It is self evident that the outer fibril boundary is longer than the inner one and hence should correspond to more band periods than the latter. In the case of a sharp kink, where the bands themselves remain undistorted, i.e. straight on both sides, the additional distance for a 100nm diameter fibril would be 72.8nm, just over one repeat. From Figs. 7 and 9 it is apparent that crimp sites are sharp enough to envisage their generation by the insertion of one additional period say half way across the fibril leaving the band otherwise unaffected. It further follows that two closely adjacent fibrils should be out of band register as they pass through the crimp. (These latter points were first recognized and communicated to us by Kastelic et al. (1978).) Thus in Fig. 7 there are 4, in Fig. 9 there are 2 additional bands on the left hand side of the fibril pair between the marker points (hence 1 and 2 extra bands per fibril respectively). The discontinuities at the crimp site thus involve only a fraction of the length of a tropocollagen molecule (290nm. Such discontinuity on the submolecular scale have further implications for the nature and orgin of the crimp. With the knowledge of the appearance of crimp sites cut along the axes of constituent fibrils, it is possible to detect the evidence for and location of such sites in many randomly cut sections of RTT. Figure 11 shows a low magnification electron micrograph of such bundles of fibrils where the crimp plane direction was not deliberately selected. Even so the crimp plane cannot be much inclined to the section plane in view of the large
Legendfor Paqe 76 Fig. 5. Sections cut from a block as in Fig. 4, viewed under crossed polars; tendon direction horizontal (a) with right extreme crimp arm parallel to polarizer or analyser, (b) with main fibre axis parallel to polarizer or analyser, (c) with left extreme crimp arm parallel to polarizer or analyser. All × 284.
78
J. Dlugosz. L. J. Gathercole and A. Kelkr
TEM and Polarizing Optics of Rat Tail Tendon crimp angle (possibly also enhanced by compression). It is being illustrated because it reveals a roughly periodic subdivision into crimped fibre assemblies of 21am across the fibre direction which is noteworthy as it may indicate a further structure element between the fibril and the tendon. In fact the smallest crimped fibre units which could be identified by previous polarizing microscopy in teased tendons were of this magnitude. This may well represent a basic ribbon element formed by the fibrils which must exist to impart an overall planarity to the structure as a whole (Gathercole and Keller, 1975). At this point we draw attention to the crimping sheet type units seen in prenatal tendon by scanning microscopy (Gathercole and Keller, 1978b) which are likely to correspond to these units. A higher magnification picture revealing an oblique cut closer to the crimp site is shown in Fig. 12. Fibrils have been cut at different inclinations depending on their location relative to the site. The feature arrowed represents the most readily visualizable situation. Here we see fibril cross-sections at the ends of the bundle and longitudinal sections centrally in between them. We interpret this as the section plane being perpendicular to the crimp plane (i.e. we see the crimp plane edge-on) with the section itself passing through the crimp region.
I
Fig. 14. Diagram showing the origin of the change in banding direction in oblique cross-sections of fibrils traversing a crimp site. Striations indicate the 68nm banding. In a thin section only the striated elipses will be visible of the truncated kinked fibril. Almost a perfect cross section of fibrils traversing a crimp is shown in Fig. 13. Most noticeable is the banding across the elliptical cross-section of the fibrils. The bands correspond to projections of the 68nm period revealed
79
by oblique cutting through more than one period, and signify (Parry and Craig, 1977) that the 68nm banding is a disc traversing the full fibril and not a surface feature. The direction of the bands and its changes across the dashed line is also informative. Figure 14 shows the expected appearance of the banding pattern of a fibril cut in cross-section as it passes through a sharp crimp plane, perpendicular to the paper surface. From this it is apparent that it is possible to follow the site by detecting the --,90 ° change in direction of the projection of the 68nm banding pattern that occurs within the elliptical fibril cross sections. At some points it is possible to detect this change within the individual fibrils themselves in cases where the section traverses the crimp itself. The sections through such fibrils have the characteristic bent shape of two ellipses overlapping at one end of their major axes. They show an internal change in banding direction (arrow) in Fig. 13 (see also Parry and Craig, 1977). It is thus apparent that crimp sites are now detectable in longitudinal sections of RTT cut at any rotational angle along the unit fibre axis, from the position in Figs. 6-9 where the constituent fibrils are exposed in perfect longitudinal section, to that in Fig. 13, revealing them in cross section. This complete difference of appearance again emphasises the planar nature of crimps. CONCLUSIONS These studies serve to demonstrate the basic correctness of the original model of the microscopic waveforms in RTT by extending our knowledge on crimping to the fibril level. By sectioning in a variety of angles, the planar nature of crimps is again confirmed. Most noteworthy is the demonstrated existence of sharp angular crimp originally proposed as a mathematical convenience (Diamant et al., 1972) without direct evidence. We now see that even such details of the original model correspond to physical reality. The accurate location of the crimp site on the electron microscopic scale opens up the possibility for description and interpretation of the crimp in fine structural and even molecular terms.
Legend for Page 78 Fig. 6. Low magnification electron micrograph of a section cut from the same block as Fig. 5, showing a single crimp site. Knife movement direction along one of the crimp arms (arrow). x 5500. Fig. 7. Higher magnification electron micrograph of crimp site as in Fig. 6. x 20,600.
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J. Dlugosz, L. J. Gathercole and A. Keller
TEM and Polarizing Optics of Rat Tail Tendon
REFERENCES Baer, E., Gathercole, L. J. and Keller, A., 1975. Structure hierarchies in tendon collagen: an interim summary. In: Structure of Fibrous Biopolymers, Colston Papers No. 26, Atkins, E. D. T. and Keller, A. (eds.), Butterworths, London, pp. 189-196. Diamant, J., Keller, A., Baer, E., Litt, M. and Arridge, R. G. C., 1972. Collagen: ultrastructure and its relation to mechanical properties as a function of ageing. Proc. R. Soc. Lond. B. 180: 293-315. Gathercole, L. J., Keller, A. and Shah, J. S., 1974. The periodic wave pattern in native tendon collagen: correlation of polarizing with scanning electron microscopy. J. Microsc., 102: 95-106. Gathercole, L. J. and Keller, A., 1975. Light microscopic waveforms in collagenous tissues and their structural implications. In: Structure of Fibrous Biopolymers, Colston Papers No. 26, Atkins, E. D. T. and Keller, A. (eds.), Butterworths, London, pp. 153-187. Gathercole, L. J. and Keller, A., 1978a. X-ray diffraction effects related to superstructure in rat tail tendon
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collagen. Biochim. biophys. Acta (in press.). Gathercole, L. J., Booy, F. P., Dlugosz, J. and Keller, A. 1978. Low temperature electron diffraction and beam effects in tendon collagen. Connective Tissue Res. 5: (In press.) Gathercole, L. J. and Keller, A., 1978b. Early development of crimping in rat tail tendon collagen: a polarizing optical and SEM study. Micron, 9: 83-89. Kastelic, J., Galeski, A. and Baer, E., 1978. The multicomposite structure of tendon. Connective Tissue Res. (in press). Parry, D. A. D. and Craig, A. S., 1977. Quantitative electron microscopic observation of the collagen fibrils in rat tail tendon. Biopolymers, 16: 1015-1031. Spurr, A. R., 1969. A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res., 26: 31-43. Torp, S., Arridge, R. G. C., Armeniades, C. D. and Baer, E., 1975. Structure-property relationships in tendon as a function of age. In: Structure of Fibrous Biopolymers, Colston Papers No. 26. Atkins, E. D. T. and Keller, A. (eds.), Butterworths, London, pp. 197-221.
Legend for Page 80 Fig. 8. Low magnification electron micrograph of a crimp site as in Fig. 6, but cut with knife direction bisecting the crimp angle (arrow). x 7700. Fig. 9. Higher magnification electron micrograph of a crimp site as in Fig. 7. x 32,000.
Legend for Page 82 Fig. 1I. Low magnification electron micrograph of section with crimp plane not selected, x 2960. Fig. 12. Electron micrograph of obliquely cut section close to a crimp, viewing the crimp plane edge-on. x 3800. Fig. 13. Higher magnification electron micrograph showing fibrils cut in cross section while traversing a crimp site. Location of the crimp site is indicated by the dashed line. Typical bent fibrils cut on the site are also indicated, x 26,200.
82
J. Dlugosz, k. J. Gathercole and A. Keller
0047-7206/78/0601-0071 $02.00/0
Micron
TRANSMISSION ELECTRON MICROSCOPE STUDIES AND THEIR RELATION TO POLARIZING OPTICAL MICROSCOPY IN RAT TAIL TENDON J. DLUGOSZ, L. J. GATHERCOLE AND A. KELLER H. H. Wills Physics Laboratory, University of Bristol, Tyndall Avenue, Bristol BS8 1TL, U.K.
(Received 17 November 1977; received for publication 20 January 1978)
Abstract--An investigation on rat tail tendon is described which follows the periodic banding pattern seen by the polarizing light microscope down to the level of transmission electron microscopy. This work was carried out by longitudinal sectioning of preselected tendon portions in preselected orientations around the tendon axis. In the course of this, full bridging of light with electronmicroscopic structural information has been achieved. In particular, complete verification is provided for the planar crimp structure as originally postulated from polarizing microscopy. "l?hus thin sections fully conformed even to the idealized crimp representation when viewed under the polarizing microscope, while the crimp sites themselves could be detected. These were seen to be sharp as implied by the rigid hinge model, which was invoked previously merely as a mathematical convenience to account for the mechanical behaviour. This sharpness of the crimp site persisted down to the level of the 68rim banding visible under the electron microscope and was seen to occur within 1-2 such band periods. Various individual features seen at all levels are discussed.
INTRODUCTION The main features of crimping in collagenous fibres of extensible connective tissues have been elucidated by ourselves and co-workers (Diamant et al., 1972; Gathercole and Keller, 1975; Baer et al., 1975; Torp et al., 1975). The aim of the present paper is to reveal the crimp sites by transmission electron microscopy (TEM) in order to confirm their existence directly, to obtain information on their nature and thus open up the possibility of studies including possibly electron diffraction (Gathercole et al., 1978a). If realized, this would complet ethe continuity of information gained from polarizing microscopy (Diamant et al., 1972) X-ray diffraction (Gathercole and Keller, 1975; 1978a)and scanning electron microscopy (Gathercole et al., 1974) from the fibre to the fibril level in rat tail tendon (RTT). As will be apparent, this has been achieved.
without teasing and cut into 5mm lengths. These were fixed in standard cacodylate-buffered 12% aqueous glutaraldehyde pH 7 at room temperature for 20min. Dehydration was in successive changes of 33%, 66% and 100% ethanol with final permeation in Spurr (1969) resin monomer. Samples were then permeated with Spurr reactive mixture and polymerized. Embedded samples were examined by polarizing microscopy to check for the retention of crimp and to determine the orientation of the crimp plane locally in the areas it was intended to cut. Sections were trimmed and then cut on an LKB Ultramicrotome, with continuous visual monitoring and adjustment to ensure cutting was parallel to the crimp plane. Sections were cut a few at a time, then examined under crossed polars to check the crimp plane alignment. Some sections were also cut at random angles to the local crimp plane. Sections were cut with knife movement both parallel and perpendicular to the bulk fibre axis in order to investigate the influence of cutting direction on the shape of the crimp site.
EXPERIMENTAL Tendon units of 6-month old rats were released from surrounding tissue in the tail 71
72
J. Dlugosz, L. J. Gathercole and A. Keller
TEM and Polarizing Optics of Rat Tail Tendon Sections were stained with 1 ~ aqueous uranyl acetate, and examined in a Philips 301 electron microscope. Some sectioning was carried out also with the section plane perpendicular to the fibre axis but because the corresponding information, plus more, was also contained in some random longitudinal sections (namely where crimp plane is perpendicular to the section plane) this will not be illustrated separately. RESULTS AND DISCUSSION Optical microscopy Figure 1 shows the block from which sections were cut containing a unit fibre of RTT with crimp plane lying in the section plane. This corresponds to the p -- 0 ° position of Gathercole and Keller (1978a) viewed in incident light. In this case, illumination is from above along the fibre axis at an angle of about 40 ° to the fibre. The appearance is surprisingly similar to that obtained with transmitted polarizing optics with one arm of the crimp parallel to polarizer or analyser (see for instance Diamant et al., 1972). These are transverse bright and dark bands due to specular reflections from surfaces composed of bundles of parallel fibres sharply bending into and out of reflecting position. These bands lie within and throughout the fibre, and their contrast can be made to reverse by rotating the illumination direction through 180 ° with respect to the fibre. The optical behaviour of this tendon shows a close resemblance to that displayed by the semiprecious gemstone 'tiger's eye' most strikingly displayed, when, as we have done, the two are viewed side-by-side. It is known that, in this mineral, sheets of parallel fibres bend sharply in varying directions, giving rise to lustrous cross bandings of brown-gold which move with changes in viewing direction (chatoyancy). This too is a fibre plus matrix system, the kinked fibres of crocidolite being embedded in a matrix of silica, thus providing a documented example of the phenomenon we are observing in our tendons.
73
Figure 2 shows a specimen block viewed in combined transmitted and incident ordinary light, from which about half the fibre has been removed by stripping. Here the crimp sites are seen directly. The sites show visually that there exists a continuous gradation in sharpness of such sites. Similarly, sites originating as very sharp on, say, the extreme left hand side of the fibre at A may become smoother as they traverse the fibre to the opposite edge B. In this, the pictures are in agreement with an earlier SEM picture invoked in support of the interpretation of complex features in the low angle X-ray patterns related to the 68nm periodicity (Gathercole and Keller, 1978a). A similar specimen is seen in Fig. 3 viewed in transmitted ordinary light. Here crimp sites are seen directly, though there is an indication of complexity in that there is evidence of an underlying reversal in the direction of the fibrillar texture at crimp sites. Such reversals are indicated by arrows (see also Fig. 4). A similar specimen is seen in Fig. 4, viewed in transmitted light with crossed polars. Figure 4a shows the specimen with the bulk fibre axis parallel to the polarizer or analyser while Figs. 4b and 4c show the stage rotated so that alternate crimp arms are parallel to the polarizer or analyser. This shows some differences from the behaviour expected from the simple model of Diamant et al. (1972). In the first instance, broad dark bands are not seen on rotation of the stage by 0o; instead, alternate crimp sites, originally dark, become bright, though their position is still obvious. Similarly, there appears to be a more deeply situated fibrillar texture running some half crimp wavelength out of phase with that obvious on the surface. Such features have first been noted in glutaraldehyde fixed adult specimens soaked in hexylene glycol (Kastelic et al., 1978) and subsequently also in fresh, wet 3-week old specimens (Gathercole and Keller, 1978b). It is interesting to note, however, that in Fig. 4, the crimp sites are fully traceable to the edge of the tendon unit and that at the edges there is no uniform appearance of flattening
Legend for Paoe 72 Fig. 1. An embedded RTT unit fibre viewed in incident light showing marked chatoyancy of the crimps. Direction of illumination is down the fibre along the axis at ,~40 ° into the fibre, x 100. Fig. 2. An embedded RTT unit fibre from which about half has been stripped. Viewed with combined transmitted and incident light x 100. Fig. 3. Enlarged view of a fully embedded RTT in transmitted light showing crimp sites. The crimp is out of phase in the lower half of the tendon; this is seen diffuselyand is indicated by the marker arrows on the left. x 370.
74
.I. Dlugosz, L. J. Gathercole and A. Keller
J
TEM and Polarizing Optics of Rat Tail Tendon out of the crimp--an observation of some relevance to current considerations (Kastelic et al. 1978) of how the crimped unit may constitute the tendon (a point not to be pursued here). All this is in marked contrast to the appearance of sections taken from these blocks when such sections are viewed in the polarizing microscope. Sections for this purpose were cut to a thickness of about llam on the Ultramicrotome. Figure 5 shows the appearance of longitudinal sections with cutting direction perpendicular to the fibre axis. Figure 5a shows the appearance with one arm of the crimp parallel to the polarizer or analyser, Fig. 5b the median position, with only the very narrow crimp sites themselves in the extinguishing position, while Fig. 5c shows the alternate arms of the crimp parallel to the polarizer or analyser direction. As can be seen, the simple behaviour of Diamant et al. (1972) is followed. The bands change from narrow to broad on stage rotation, the crimp sites are very sharp and the planarity of the crimp is self evident. Clearly, whatever the packing on a larger scale it is possible from these pictures to obtain coherent sheets of planar crimping fibrils that extend over the whole width of the unit fibre. Sections were also cut parallel to the main fibre axis. The general appearance was the same as the specimens cut perpendicular to the axis, except that the crimp is further compressed. In practice, it was found to be easier to obtain coherent longitudinal sections with the knife edge moving perpendicular to the fibre axis. Electron microscopy The sites displayed as in Fig. 5 can be viewed directly in the electron microscope. Figure 6 shows a low magnification view of an ultrathin section cut from the same block as in Fig. 5. There is a sharp kink within each adjacent fibril and alignment between adjacent kinks. The cutting direction is as shown, approximately parallel to the lower arm of the crimp. Figure 8 shows a similar area where the crimp has an overall smooth appearance. Here cutting direction very nearly bisects the angle enclosed by the two crimp arms and this may well have contributed to a 'flattening' of crimp with a corresponding
75
smoothing out of the discontinuity at the crimp site. The crimps in individual fibrils are still sharp, though these deflect through a smaller angle and are less well aligned, contributing to an average smoothness. Higher enlargement micrographs (Figs. 7 and 9 respectively) readily reveal the striations corresponding to the 68nm periodicity familiar from the fine structural electron microscopy of collagen. The main points to note are as follows. First, the existence of the crimped structure has now been demonstrated by transmission electron microscopy. Second, that the crimped arrangement is definitely planar otherwise the individual fibres could not be seen along any appreciable length within a given section (in fact they are seen to extend over the full section) and third, that the crimp is indeed sharp. All these features were previously deduced from polarizing optics of the full tendon units (Diamant et al., 1972; Gathercole and Keller, 1975) and/or inferred (the sharp hinge of the crimp) from the mechanical model constructed to account for its mechanical properties. Fourth, the dimensional level has been reached where the 68nm period has become visible simultaneously with the crimp site. We see therefore that the present work successfully extends the previous low power microscopic observations and the inferences from the mechanical behaviour to the level of the electron microscope. This establishes complete continuity between the macroscopic, microscopic and ultrafine structure research methods transferring the results of the former to the latter level. Examination of the 68nm bandingyields further information. The bands are skewed with respect to the fibril axis and this is in different directions in the corresponding crimp arms for parallel and perpendicular cutting (Figs. 7, 9). Also for a given cutting direction this skewing is of opposed direction in the two crimp arms (all these effects are shown schematically in Fig. 10). All this indicates that the skewing was largely induced by the cutting, although the existence of much smaller amounts of skewing along the crimp arm parallel to the cutting direction (Fig. 7) suggests that some obliquity may have also been present
Leyend for Paye 74 Fig. 4. Same specimen as in Fig. 3 viewed in transmitted light with crossed polars, (a) with the bulk fibre axis parallel to analyser or polarizer, (b) and (c) with alternate crimp arms parallel to polarizer or analyser. All x 190.
76
J. Dlugosz~ L. J. Gathercole and A. Keller
T E M and Polarizing Optics of Rat Tail Tendon CRIMP SITE
C
FIBRIL
(Q)
~
CRIMP SITE
FIBRIL.
Ib) CUT
Fig. 10. Diagram showing the origin of shear effects in crimp compression and 68nm band tilting as shown in Figs. 6 and 7 (10a) and 8 and 9 (10b). The central region shows a single fibril within a suggested array of parallel fibrils (not to scale).
ab initio in the native tendon (the possibility of this latter effect has been raised in connection with our earlier X-ray work (Gathercole and Keller, 1978a)). Concentrating on the more pronounced effects which are definitely induced by the cutting process, we note that the shear is such that the 68nm bands tend to align with the edge of the knife. Thus in Fig. 7 they become almost parallel to the line containing the crimp site while in Fig. 9 they tilt in the opposite direction. This is shown simplified in Fig. 10. We can consider this as resulting from shear deformation on compression, the direction of which is along the movement of the knife. It will be seen that if planes of easy slip exist along the fibril direction then such slip, as activated by the compression, would produce the observed direction of skewing of the bands in all cases. Slippage along the direction of the tropocoUagen molecule is of course reasonable, although the readiness by
77
which this takes place may be noteworthy. It is important, however, to note that the preservation of the banding on such sqppage implies that the units which slide past each other already possess the 68nm periodicity. Thus it cannot be on the scale of the triple helix itself but it must involve an appropriate multiple of it such as already embodies the quarter stagger believed to be responsible for the 68nm banding. A point to the same effect has already been made previously (Gathercole and Keller, 1978a). Observations can also be made concerning the register and numbering of 68nm bands across crimp sites. It is self evident that the outer fibril boundary is longer than the inner one and hence should correspond to more band periods than the latter. In the case of a sharp kink, where the bands themselves remain undistorted, i.e. straight on both sides, the additional distance for a 100nm diameter fibril would be 72.8nm, just over one repeat. From Figs. 7 and 9 it is apparent that crimp sites are sharp enough to envisage their generation by the insertion of one additional period say half way across the fibril leaving the band otherwise unaffected. It further follows that two closely adjacent fibrils should be out of band register as they pass through the crimp. (These latter points were first recognized and communicated to us by Kastelic et al. (1978).) Thus in Fig. 7 there are 4, in Fig. 9 there are 2 additional bands on the left hand side of the fibril pair between the marker points (hence 1 and 2 extra bands per fibril respectively). The discontinuities at the crimp site thus involve only a fraction of the length of a tropocollagen molecule (290nm. Such discontinuity on the submolecular scale have further implications for the nature and orgin of the crimp. With the knowledge of the appearance of crimp sites cut along the axes of constituent fibrils, it is possible to detect the evidence for and location of such sites in many randomly cut sections of RTT. Figure 11 shows a low magnification electron micrograph of such bundles of fibrils where the crimp plane direction was not deliberately selected. Even so the crimp plane cannot be much inclined to the section plane in view of the large
Legendfor Paqe 76 Fig. 5. Sections cut from a block as in Fig. 4, viewed under crossed polars; tendon direction horizontal (a) with right extreme crimp arm parallel to polarizer or analyser, (b) with main fibre axis parallel to polarizer or analyser, (c) with left extreme crimp arm parallel to polarizer or analyser. All × 284.
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J. Dlugosz. L. J. Gathercole and A. Kelkr
TEM and Polarizing Optics of Rat Tail Tendon crimp angle (possibly also enhanced by compression). It is being illustrated because it reveals a roughly periodic subdivision into crimped fibre assemblies of 21am across the fibre direction which is noteworthy as it may indicate a further structure element between the fibril and the tendon. In fact the smallest crimped fibre units which could be identified by previous polarizing microscopy in teased tendons were of this magnitude. This may well represent a basic ribbon element formed by the fibrils which must exist to impart an overall planarity to the structure as a whole (Gathercole and Keller, 1975). At this point we draw attention to the crimping sheet type units seen in prenatal tendon by scanning microscopy (Gathercole and Keller, 1978b) which are likely to correspond to these units. A higher magnification picture revealing an oblique cut closer to the crimp site is shown in Fig. 12. Fibrils have been cut at different inclinations depending on their location relative to the site. The feature arrowed represents the most readily visualizable situation. Here we see fibril cross-sections at the ends of the bundle and longitudinal sections centrally in between them. We interpret this as the section plane being perpendicular to the crimp plane (i.e. we see the crimp plane edge-on) with the section itself passing through the crimp region.
I
Fig. 14. Diagram showing the origin of the change in banding direction in oblique cross-sections of fibrils traversing a crimp site. Striations indicate the 68nm banding. In a thin section only the striated elipses will be visible of the truncated kinked fibril. Almost a perfect cross section of fibrils traversing a crimp is shown in Fig. 13. Most noticeable is the banding across the elliptical cross-section of the fibrils. The bands correspond to projections of the 68nm period revealed
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by oblique cutting through more than one period, and signify (Parry and Craig, 1977) that the 68nm banding is a disc traversing the full fibril and not a surface feature. The direction of the bands and its changes across the dashed line is also informative. Figure 14 shows the expected appearance of the banding pattern of a fibril cut in cross-section as it passes through a sharp crimp plane, perpendicular to the paper surface. From this it is apparent that it is possible to follow the site by detecting the --,90 ° change in direction of the projection of the 68nm banding pattern that occurs within the elliptical fibril cross sections. At some points it is possible to detect this change within the individual fibrils themselves in cases where the section traverses the crimp itself. The sections through such fibrils have the characteristic bent shape of two ellipses overlapping at one end of their major axes. They show an internal change in banding direction (arrow) in Fig. 13 (see also Parry and Craig, 1977). It is thus apparent that crimp sites are now detectable in longitudinal sections of RTT cut at any rotational angle along the unit fibre axis, from the position in Figs. 6-9 where the constituent fibrils are exposed in perfect longitudinal section, to that in Fig. 13, revealing them in cross section. This complete difference of appearance again emphasises the planar nature of crimps. CONCLUSIONS These studies serve to demonstrate the basic correctness of the original model of the microscopic waveforms in RTT by extending our knowledge on crimping to the fibril level. By sectioning in a variety of angles, the planar nature of crimps is again confirmed. Most noteworthy is the demonstrated existence of sharp angular crimp originally proposed as a mathematical convenience (Diamant et al., 1972) without direct evidence. We now see that even such details of the original model correspond to physical reality. The accurate location of the crimp site on the electron microscopic scale opens up the possibility for description and interpretation of the crimp in fine structural and even molecular terms.
Legend for Page 78 Fig. 6. Low magnification electron micrograph of a section cut from the same block as Fig. 5, showing a single crimp site. Knife movement direction along one of the crimp arms (arrow). x 5500. Fig. 7. Higher magnification electron micrograph of crimp site as in Fig. 6. x 20,600.
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TEM and Polarizing Optics of Rat Tail Tendon
REFERENCES Baer, E., Gathercole, L. J. and Keller, A., 1975. Structure hierarchies in tendon collagen: an interim summary. In: Structure of Fibrous Biopolymers, Colston Papers No. 26, Atkins, E. D. T. and Keller, A. (eds.), Butterworths, London, pp. 189-196. Diamant, J., Keller, A., Baer, E., Litt, M. and Arridge, R. G. C., 1972. Collagen: ultrastructure and its relation to mechanical properties as a function of ageing. Proc. R. Soc. Lond. B. 180: 293-315. Gathercole, L. J., Keller, A. and Shah, J. S., 1974. The periodic wave pattern in native tendon collagen: correlation of polarizing with scanning electron microscopy. J. Microsc., 102: 95-106. Gathercole, L. J. and Keller, A., 1975. Light microscopic waveforms in collagenous tissues and their structural implications. In: Structure of Fibrous Biopolymers, Colston Papers No. 26, Atkins, E. D. T. and Keller, A. (eds.), Butterworths, London, pp. 153-187. Gathercole, L. J. and Keller, A., 1978a. X-ray diffraction effects related to superstructure in rat tail tendon
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collagen. Biochim. biophys. Acta (in press.). Gathercole, L. J., Booy, F. P., Dlugosz, J. and Keller, A. 1978. Low temperature electron diffraction and beam effects in tendon collagen. Connective Tissue Res. 5: (In press.) Gathercole, L. J. and Keller, A., 1978b. Early development of crimping in rat tail tendon collagen: a polarizing optical and SEM study. Micron, 9: 83-89. Kastelic, J., Galeski, A. and Baer, E., 1978. The multicomposite structure of tendon. Connective Tissue Res. (in press). Parry, D. A. D. and Craig, A. S., 1977. Quantitative electron microscopic observation of the collagen fibrils in rat tail tendon. Biopolymers, 16: 1015-1031. Spurr, A. R., 1969. A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res., 26: 31-43. Torp, S., Arridge, R. G. C., Armeniades, C. D. and Baer, E., 1975. Structure-property relationships in tendon as a function of age. In: Structure of Fibrous Biopolymers, Colston Papers No. 26. Atkins, E. D. T. and Keller, A. (eds.), Butterworths, London, pp. 197-221.
Legend for Page 80 Fig. 8. Low magnification electron micrograph of a crimp site as in Fig. 6, but cut with knife direction bisecting the crimp angle (arrow). x 7700. Fig. 9. Higher magnification electron micrograph of a crimp site as in Fig. 7. x 32,000.
Legend for Page 82 Fig. 1I. Low magnification electron micrograph of section with crimp plane not selected, x 2960. Fig. 12. Electron micrograph of obliquely cut section close to a crimp, viewing the crimp plane edge-on. x 3800. Fig. 13. Higher magnification electron micrograph showing fibrils cut in cross section while traversing a crimp site. Location of the crimp site is indicated by the dashed line. Typical bent fibrils cut on the site are also indicated, x 26,200.
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J. Dlugosz, k. J. Gathercole and A. Keller