School of Geography and Earth Sciences, McMaster University, Hamilton, ON, L8S 4K1, Canada.
School of Biomedical Engineering, McMaster University, Hamilton, ON, L8S 4L7, Canada.
J Bone Miner Metab. 2024 May;42(3):344-351. doi: 10.1007/s00774-024-01507-5. Epub 2024 May 11.
Transmission electron microscopy (TEM) is widely used to study the ultrastructure of bone. The mineral of bone occurs as polycrystalline mineral plates about 3 to 6 nm in thickness. A problem in using TEM to make quantitative analyses of bone is that the orientation of the plates with respect to the plane of the section being imaged is expected to affect their apparent thickness. The purpose of this study was to test if this was true, if the apparent thickness of plates changed substantially as a result of tilt of the section.
We prepared TEM sections of samples of cortical human bone by ion beam milling, orienting one section parallel to the collagen fibril axes and one perpendicular to them. We obtained TEM bright field and HAADF images of these sections, tilting the sections up to ± 20° at 2° intervals and measuring the apparent thickness of individual mineral platelets at each angle of tilt.
Thickness appears to double as section is tilted ± 20°. True thickness of plates is determined by tilting the section along an axis parallel to the plate orientation and determining the minimum apparent thickness. However, as plates are tilted away from minimum-thickness orientation, they become less well-resolved, disappearing when tilted more than 20°. We therefore also measured apparent thickness of only the darkest (most electron scattering) plate images in an untilted section and obtained the same average thickness as that obtained by tilting.
We conclude that tilting of the section is not necessary to obtain an accurate measurement of the thickness of mineral plates.
透射电子显微镜（TEM）广泛用于研究骨的超微结构。骨的矿物质以约 3 至 6nm 厚的多晶矿物质板的形式存在。在使用 TEM 对骨进行定量分析时，存在一个问题，即板相对于被成像切片平面的方向预计会影响其表观厚度。本研究的目的是测试这种情况是否属实，即由于切片的倾斜，板的表观厚度是否会发生实质性变化。
我们通过离子束铣削制备了皮质人骨样本的 TEM 切片，使一个切片与胶原纤维轴平行，另一个切片与之垂直。我们获得了这些切片的 TEM 明场和 HAADF 图像，将切片倾斜至±20°，每隔 2°测量一次各个矿物质板的表观厚度。
当切片倾斜±20°时，厚度似乎翻倍。板的真实厚度通过沿与板取向平行的轴倾斜切片并确定最小表观厚度来确定。然而，随着板从最小厚度取向倾斜，它们变得不太清晰，当倾斜超过 20°时就会消失。因此，我们还测量了未倾斜切片中仅最暗（最能散射电子）板图像的表观厚度，并获得了与倾斜测量相同的平均厚度。
我们得出结论，为了准确测量矿物质板的厚度，不需要倾斜切片。
