High resolution SEM should be, in theory, the most useful instrument to investigate the extracellular matrix of connective tissues, in that it should provide information on the molecular arrangement at 3D level. In SEM, however, collagen fibrils are hardly distinguishable from the other components. In fact while in TEM the collagen fibril is easily detectable for its periodicity, in SEM the latter is rarely seen (1). This may be due to the conductive coating that usually masks the collagen banding.In this work we tried to develop coating method, that allow the visualization of collagen period.
Methods
Unfixed mouse tail tendons were quickly dissected in filaments as thin as possible. Tissues were dehydrated in a graded acetone series, dried for two hours in high vacuum. They were then sputtered with 26 nm gold palladium (Polaron 5001 S.D.), or with 18 nm chromium (Plasma science Cr C 100 S.D.), or rotary shadowed with 2 m platinum /carbon (Baker BAF 301, two EK 552 E.B.G.), at -85. All instruments were equipped with film thickness monitors. Specimens were kept overnight in high vacuum and observed with a field emission Hitachi S 4000 SEM, at 20 kV.
Results
Collagen period had a length of 64 nm in all specimens. At high resolution, in specimens coated with gold palladium, brightness and contrast were good. However particles of various dimensions were evident on the fibrils and the collagen period was partially masked with the thick metal covering. Fractures were evident: some of them, perpendicular to the collagen fibrils axis, were spaced each other a period length; others, parallel to the axis, rarely exceeded the collagen period length (Fig. 1A). In chromium-coated specimens, brightness and contrast were not satisfactory, where the covering was rather homogeneous. The period was evident and collagen fibrils gap and overlap zones were easily observable. In addition, in these specimens, fractures absolutely comparable with those observed with gold palladium were seen (Fig. 1B). In platinum/carbon-coated specimens, the brightness was very satisfactory and the covery homogeneous. Period was easily visible and gap and overlap zones were evident. The gap zone represented about 1/5 of the total period. No fracture could be seen in these specimens (Fig. 1C). At a relatively low magnification, the periodicity of the fibrils could be appreciated (Fig. 2). DISCUSSION Unfixed fragments were used in order to avoid artefacts produced by furation with aldehydes and tannic acid (both create artificial cross-links between collagen molecules) (2). From our results it emerges that gold palladium covering gives unsatisfactory results for its granularity. Collagen period was easily observable after chromium coating: gap and overlap zones beeing well distinguishable. Fractures observed in gold and chromium-covered specimens suggestively recall the arrangement of proteoglycans as results from TEM (3). Even though no subperiod was observed, platinudcarbon coating gave the best results because it allowed the identification of gap and overlap zones. These fidings might be related more to the coating temperature than to the metal used: according to Hermann and Muller (5,6) the metal grain size increases with heating. At variance with TEM findings, that showed a period of 67 nm and a gap zone representing 2/5 (4), according to our results the period measures 64 nm and gap zone only 1/5 of the total period. Even though several other factors may be taken into consideration, we believe that the shortening of the period and of the gap zone observed in SEM may be related to the fact that we employed unfixed tissues.