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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.^[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.^[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 atThis requires 100 inter-image cuts. If a data format assigns each face a $40\mathrm{nm}$-deep voxel, its nominal represented thickness is instead $N\Delta z=4.04\mu \mathrm{m}$. 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.