Stereological techniques using the optical disectors require estimation of final section thickness, but frozen tissue irregularities may interfere with this estimation. Cryostat slices from rodent nerve tissues (dorsal root ganglia, spinal cord, and brain), cut at 16, 40, and 50 μm, were digitized with a confocal microscope and visualized through 3D software. Geometric section thickness of tissue (Tgeom) was defined as tissue volume/area. Maximal section thicknesses (Tmax), from the top to the bottom of the section, were measured in a random sample of vertical ZX planes. Irregularities were mostly related to blood vessels traversing the tissue and neuronal somas protruding over the cut surfaces, with other neuron profiles showing a fragmented appearance. Irregularities contributed to increasing the distance between the tops and bottoms of slices sectioned in different laboratories. Significant differences were found between Tmax and Tgeom for all thickness studies and counting frames (p<0.01). The Tgeom/Tmax average rate was 68.4–85.7% in volumes around cell profiles (∼600–1,200 μm2) and 83.3–91.8% in subcellular samples (∼25–160 μm2). Confocal microscopy may help to assess tissue irregularities, which might lead to an overestimation of tissue volume if section thickness is estimated by focusing on the top and bottom of the sections.

One of the difficulties in analyzing atomic resolution electron microscope images is that the sample thickness is usually unknown or has to be fitted from parameters that are not precisely known. An accurate measure of thickness, ideally on a column-by-column basis, parameter free, and with single atom accuracy, would be of great value for many applications, such as matching to simulations. Here we propose such a quantification method for annular dark field scanning transmission electron microscopy by using the single electron intensity level of the detector. This method has the advantage that we can routinely quantify annular dark field images operating at both low and high beam currents, and under high dynamic range conditions, which is useful for the quantification of ultra-thin or light-element materials. To facilitate atom counting at the atomic scale we use the mean intensity in an annular dark field image averaged over a primitive cell, with no free parameters to be fitted. To illustrate the potential of our method, we demonstrate counting the number of Al (or N) atoms in a wurtzite-type aluminum nitride single crystal at each primitive cell over the range of 3–99 atoms.

An accurate determination of specimen thickness is essential for quantitative analytical electron microscopy. Here we demonstrate that a position-averaged incoherent bright-field signal recorded on an absolute scale can be used to determine the thickness of on-axis crystals with a precision of ±1.6 nm. This method measures both the crystalline and the noncrystalline parts (surface amorphous layers) of the sample. However, it avoids the systematic error resulting from surface plasmon contributions to the inelastic mean-free-path thickness estimated by electron energy loss spectroscopy.