Atomic Force Microscopy Protocol for Collagen Morphology

Following AFMindentation, the polished surface was partially decalcified by soaking the bones in 0.5 M EDTA for 25 minutes followed by five minutes of sonication in a water bath. This process was repeated five times for each sample. For imaging, RTESPA probes were used (Bruker; radius nominally 8 nm, spring constant  = 40 N/m). The scan size was set at 3.5 µm with 512×512 pixels and a scan rate of 0.5 lines/s. For measurements of collagen morphology, four locations were imaged per sample, and 10 to 15 fibrils were measured at each location. 40 to 50 fibrils per sample were averaged to produce a single value for each animal, though the individual tests were used for distribution comparisons. Using SPIP 5.1.10 (Image Metrology, H∅rsholm, Denmark),D-periodic spacing was calculated using2D Fast Fourier Transformations (2D FFTs) as previously described [28] (link).