Serial block-face scanning electron microscopy

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.

Lateral position jitter is the fluctuating in-plane displacement of successive acquired images relative to a chosen reference. In serial block-face scanning electron microscopy, it can displace corresponding features between block faces even when the cutting increment is perfectly regular. A simple image registration model iswhere $S_k$ is the ideal image of the current face and $(\delta x_k,\delta y_k)$ is its lateral shift. The specimen itself changes between faces, so not every apparent shift is instrumental motion.

A primary apparatus study estimated inter-image displacement from cross-correlation peaks in several subregions. Its Figure4 central cluster had standard deviations of $10.9\mathrm{nm}$ and $11.8\mathrm{nm}$ along the two axes; outliers and changing specimen patterns required interpretation.^[ref]

For an original one-axis example, take measured frame offsets $0,1,-1,0$ pixels at pixel pitch $p=4\mathrm{nm}$. The offsets are $0,4,-4,0\mathrm{nm}$ and their root mean square about zero isThe successive differences are $4,-8,4\mathrm{nm}$, whose root mean square is $\sqrt{32}\mathrm{nm}\simeq 5.66\mathrm{nm}$. These are different statistics. For independent equal-variance frame offsets, the theoretical difference variance is $2{\sigma }^2$; correlation or drift changes that relation. Specify the reference, detrending and normalization when reporting jitter. Image registration can estimate a correction but does not prove that all residual motion or specimen deformation has been removed.