Bright-field transmission electron microscope (TEM) images of ion milled or focused ion beam (FIB) sections of cortical bone sectioned parallel to the long axis of collagen fibrils display an electron-dense phase in the gap zones of the fibrils, as well as elongated plates (termed mineral lamellae) comprised of apatite crystals, which surround and lie between the fibrils. Energy dispersive X-ray spectroscopy (EDS) and electron energy loss spectroscopy (EELS) studies by others have shown that the material in the gap zones is calcium phosphate. Dark-field (DF) images are capable of revealing the projected position of crystals of apatite in a section of bone. We obtained bright field (BF) images of ion milled sections of bovine femoral cortical bone cut parallel to fibril axes (longitudinal view), and compared them with DF images obtained using the (002) apatite reflection to test a widely held theory that most of the mineral in bone resides in the gap zones. Most apatite crystals which were illuminated in DF images and which projected onto gap zones were extensions of crystals that also project onto adjacent overlap zones. However, in BF images, overlap zones do not appear to contain significant amounts of mineral, implying that the crystals imaged in DF are actually in the interfibrillar matrix but projected onto images of fibrils. However a small number of "free" illuminated crystals did not extend into the overlap zones; these could be physically located inside the gap zones. We note that projections of gap zones cover 60% of the area of any longitudinal field of view; thus these "free" crystals have a high random probability of appearing to lie on a gap zone, wherever they physically lie in the section. The evidence of this study does not support the notion that most of the mineral of bone consists of crystals in the gap zone. This study leaves uncertain what is the Ca-P containing material present in gap zones; a possible candidate material is amorphous calcium phosphate.

中文翻译：

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使用暗场照明在骨胶原纤维间隙区寻找磷灰石晶体

平行于胶原纤维长轴切片的皮质骨的离子铣削或聚焦离子束 (FIB) 切片的明场透射电子显微镜 (TEM) 图像显示纤维间隙区中的电子致密相，以及由磷灰石晶体组成的细长板（称为矿物薄片），围绕并位于原纤维之间。其他人的能量色散 X 射线光谱 (EDS) 和电子能量损失光谱 (EELS) 研究表明，间隙区中的材料是磷酸钙。暗场 (DF) 图像能够揭示磷灰石晶体在骨骼部分的投影位置。我们获得了平行于原纤维轴（纵向视图）切割的牛股骨皮质骨的离子研磨切片的明场 (BF) 图像，并将它们与使用 (002) 磷灰石反射获得的 DF 图像进行比较，以测试广泛接受的理论，即骨骼中的大部分矿物质位于间隙区。大多数在 DF 图像中被照亮并投影到间隙区域的磷灰石晶体是晶体的延伸，它们也投影到相邻的重叠区域。然而，在 BF 图像中，重叠区域似乎不包含大量矿物，这意味着在 DF 中成像的晶体实际上位于纤维间基质中，但投影到纤维图像上。然而，少数“自由”发光晶体没有延伸到重叠区；这些可以物理地位于间隙区域内。我们注意到间隙区域的投影覆盖了任何纵向视野区域的 60%；因此这些“免费” 晶体有很高的随机概率出现在间隙区域上，无论它们实际位于截面中的哪个位置。这项研究的证据不支持大多数骨骼矿物质由间隙区中的晶体组成的观点。这项研究让我们不确定间隙区中存在的含钙磷材料是什么；一种可能的候选材料是无定形磷酸钙。