Biological serial block face scanning electron microscopy at improved z-resolution based on Monte Carlo model
Biological serial block face scanning electron microscopy at improved z-resolution based on Monte Carlo model
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DOI：
10.1038/s41598-018-31231-w
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发表时间：
2018-08-28
影响因子：
4.6
通讯作者：
Leapman, R. D.
中科院分区：
文献类型：
--
作者：
He, Q.;Hsueh, M.;Leapman, R. D.
连续块面电子显微镜（SBEM）提供了包埋和染色细胞和组织的纳米级3D超微结构，体积高达10(7)μ m(3)。在SBEM中，将能量为1-3 keV的电子入射到样品块上，在块面上以5-10 nm的x， y分辨率收集背向散射电子（BSE）图像，并通过原位超微切片去除连续层。然而，由于最小切割厚度的限制，沿z方向的空间分辨率限制在25 nm左右。为了提高z分辨率，我们使用蒙特卡罗模拟电子散射，从双主光束能量下获得的BSE图像中提取深度信息。染色深度与双能BSE强度比之间的关系使我们能够以x2的z分辨率确定3D结构。我们通过肝组织中肝细胞膜的亚切片成像来证明该技术。
Serial block-face electron microscopy (SBEM) provides nanoscale 3D ultrastructure of embedded and stained cells and tissues in volumes of up to 10(7) mu m(3). In SBEM, electrons with 1-3 keV energies are incident on a specimen block, from which backscattered electron (BSE) images are collected with x, y resolution of 5-10 nm in the block-face plane, and successive layers are removed by an in situ ultramicrotome. Spatial resolution along the z-direction, however, is limited to around 25 nm by the minimum cutting thickness. To improve the z-resolution, we have extracted depth information from BSE images acquired at dual primary beam energies, using Monte Carlo simulations of electron scattering. The relationship between depth of stain and ratio of dual-energy BSE intensities enables us to determine 3D structure with a x2 improvement in z-resolution. We demonstrate the technique by sub-slice imaging of hepatocyte membranes in liver tissue.