= Serial block-face scanning electron microscopy
{wiki}
<Serial block-face scanning electron microscopy> acquires a three-dimensional dataset by repeatedly imaging an exposed specimen face in a <scanning electron microscope> and removing a thin layer with an <ultramicrotome>. The removed layer is not the imaged object: each image records the newly exposed block face. This differs from collecting and imaging thin sections in <serial-section electron microscopy>.<Serial Block Face Scanning Electron Microscopy by JEOL>[Definition, in-chamber sectioning and imaging of the newly exposed face]{ref}
In the 2004 apparatus of <Winfried Denk> and <Heinz Horstmann>, a <diamond knife> operated inside the microscope chamber, with controlled specimen advance and retraction. A <backscattered electron> signal supplied image contrast. Keeping the cutting and imaging operations in the chamber enabled automated <image stack> acquisition.<Serial Block-Face Scanning Electron Microscopy to Reconstruct Three-Dimensional Tissue Nanostructure by Winfried Denk and Heinz Horstmann>[Results, Block-Face Imaging; Materials and Methods, Microtome]{ref}
For an original ideal geometry, acquire the first face at $z=0$, then advance and remove $40\,\mathrm{nm}$ before each subsequent image. An <image stack> of $N=101$ faces has its last acquired face at
$$
z_{100}=(N-1)\Delta z=100\cdot40\,\mathrm{nm}=4.00\,\mathrm{\mu m}.
$$
This requires 100 inter-image cuts. If a data format assigns each face a $40\,\mathrm{nm}$-deep <voxel>, its nominal represented thickness is instead $N\Delta z=4.04\,\mathrm{\mu m}$. State which extent is intended. At lateral <pixel pitch> $5\,\mathrm{nm}$, the sampling ratio $\Delta z/p$ is 8.
The resulting <image stack> needs <image registration> and a calibrated cutting coordinate. <Lateral position jitter>, cutting artifacts and the depth range contributing to the electron signal limit the useful reconstruction. Finite <voxel> spacing is not itself a measurement of <spatial resolution>. Destructive cutting also means that later imaging cannot recover an exposed face that has already been removed.
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