Collagen fibril surface: TMAFM, FEG-SEM and freeze-etching observations

Native, unfixed collagen fibrils from rat tail tendon were dehydrated following different procedures and observed under a FEG-SEM and an AFM operated in Tapping Mode (TMAFM). Freeze-etched, untreated fibrils from the same tissue were also observed for comparison. The most notable features of the fibril surface, i.e., the gap/overlap alternation and three prominent intraperiod ridges, were simultaneously visible only in freeze-etched specimens, while under the SEM and the TMAFM their appearance was dependent on both the dehydration procedure and the visualization technique. The different susceptibility of the collagen fibril surface structures to various treatments clearly implies the existence of domains of different composition. Moreover, identical specimens were imaged differently by SEM and TMAFM, highlighting instrument-specific advantages and limitations. The onset of dehydration-dependent, procedure-specific artifacts should be considered in high-resolution studies of connective tissues. As for any biological specimen, the final aspect of collagen fibrils is determined no less by the preliminary treatments than by the visualization approach.