Stereology tutoral
Compared to the measured section thicknesses determined by ORE, SR measures displayed less than 1% deviation. Using a commercially available optical reflectometer (F20, Filmetrics ®, USA), the thicknesses of 0.5 μm thick semi-thin Epon (glycid ether)-sections and of 1–3 μm thick plastic sections (glycolmethacrylate/ methylmethacrylate, GMA/MMA), as regularly used in physical disector analyses, could precisely be measured within few seconds. To find a practicable, fast, and accurate alternative to ORE, the suitability of spectral reflectance (SR) measurement for determination of plastic section thicknesses was evaluated. The section angle of the re-embedded section is then calculated from the deviation of the measured section thickness of the calibration foil and its factual thickness, using basic geometry. For this, the analyzed section is mounted flat on a foil of known thickness (calibration foil) and both the section and the calibration foil are then vertically (re-)embedded. Here, an improved ORE method is presented, allowing for determination of the factual section plane angle of the re-embedded section, and correction of measured section thickness values for oblique (non-orthogonal) sectioning. However, the orthogonal re-embedding (ORE) technique is quite work- and time-intensive and may produce inaccurate section thickness measurement values due to unintentional slightly oblique (non-orthogonal) positioning of the re-embedded sample-section. The section thickness is then measured in a subsequently prepared histological section of this orthogonally re-embedded sample. One conventional method for measurement of histological section thickness is to re-embed the section of interest vertically to its original section plane. The accuracy of quantitative stereological analysis tools such as the (physical) disector method substantially depends on the precise determination of the thickness of the analyzed histological sections.