Abstract
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. One conventional method for measurement of histological section thickness is to re-embed the section of interest vertically to its original section plane. The section thickness is then measured in a subsequently prepared histological section of this orthogonally re-embedded sample. 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. 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. 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. 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. 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. 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. Compared to the measured section thicknesses determined by ORE, SR measures displayed less than 1% deviation. Our results prove the applicability of SR to efficiently provide accurate section thickness measurements as a prerequisite for reliable estimates of dependent quantitative stereological parameters.
Highlights
The thickness of a histological section generally affects the contrast, sharpness, and detail recognizability within the microscopic image of the slide
While the theoretical physical foundations and technical details of the method are described elsewhere [19, 20], spectral reflectance (SR) measurement is principally based on analysis of the pattern of reflection(s) that occur at interfaces of flat layers of different homogenous materials, when light of different wavelengths is sent through such a “thin-film” stack
Each one half was mounted on an uncoated, standard, borosilicate glass slide (ISO 8037/1, Engelbrecht Medizin und Labortechnik GmbH, Edermunde, Germany) for SR section thickness measurement, while the second section halves were mounted flat on calibration foils, orthogonally re-embedded in Epon using standard flat-embedding molds (in order to minimize the probability of unintended oblique orientations of the section-calibration foil stacks within the Epon-blocks), and re-sectioned for verification of the section thickness by light-microscopic measurement (Fig 6E)
Summary
The thickness of a histological section generally affects the contrast, sharpness, and detail recognizability within the microscopic image of the slide. These factors include e.g., the brand, quality, manufacturing standards, age, utilization rate, operational performance and service intervals of the microtome, the individual operator, the hardness of the embedding medium, the time the tissue-block was stored before sectioning, the temperature during sectioning, and the type and condition of the microtome blade In this context, it should not left unmentioned, that there are structure analysis approaches [10], as well as several quantitative stereological analysis techniques [6], including methods for estimation of numerical volume densities, which are independent of section thicknesses or embedding-related tissue shrinkage. In contrast, the embedding medium is not removed during the subsequent procession steps, and the sections display uniformly smooth surfaces and equal section thickness in areas with and without embedded tissue (Fig 2)
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