Measurement Of Section Thickness Research Articles

Practicable methods for histological section thickness measurement in quantitative stereological analyses

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.

Thick methacrylate sections devoid of lost caps simplify stereological quantifications based on the optical fractionator design.

In neuroscience, the optical fractionator technique is frequently used for unbiased cell number estimations. Although unbiased in theory, the practical application of the technique is often biased by the necessity of introducing a guard zone at one side of the disector to counter lost caps and/or optical limitations. Restricting the disector within the section thickness potentially introduces bias in two ways. First, the need to measure section thickness in order to obtain the disector height/section thickness fraction is challenging since both microcator measurements, microtome block advance, and measurements on re-embedded sections are potentially biased. Second, disector placement is not uniform random within the section thickness resulting in a bias in most sections with inhomogeneous cell distribution along the z axis. Re-embedded 2-hydroxyethylmethacrylate (hereafter methacrylate) sections were inspected for lost caps to evaluate the possibility of whole section thickness counting with the optical fractionator technique and hippocampal granular cell nucleoli density differences along the z axis were assessed with a z axis analysis. No lost caps were found in the examined re-embedded tissue and an inhomogeneous cell distribution through the section thickness was observed. In thick methacrylate sections devoid of lost caps sampling through the entire section thickness could be an acceptable alternative to the use of guard zones and the consequent biases associated with section thickness measurement and non-random placement of disectors.

Test Object for Measurement of Section Thickness at US

A simple phantom was used to test the ultrasound beam profile in the elevation direction (perpendicular to the scanning plane). Based on the inclined plane method, the phantom allowed direct clear display of the section thickness over the whole exploration depth on a single standard ultrasonographic image. This was demonstrated with one- and one and one-half-dimensional arrays. The results showed the efficiency of electronic focusing in the elevation plane associated with one and one-half-dimensional arrays.

Quantitation of gold labeling and estimation of labeling efficiency with a stereological counting method.

The amount of immunolabeling over a cell compartment of an average cell was estimated by use of an adaptation of the double disector method introduced by Gundersen. The first and last sections of a stack of ultra-thin sections formed a disector in which cell number could be estimated and related to a defined reference volume to give the cell density. Another stack section, selected at random, was immunolabeled and the number of gold particles associated with unit volume reference space (gold "density") estimated in quadrats placed systematically across the section. The ratio of gold density to cell density was used to estimate the number of gold particles lying over a chosen compartment of an average cell, N(gold)/N(cell). Such estimates required neither cell volume nor section thickness measurement and were reproducible. By combining the number of gold particles per cell with estimates of the number of protein antigens per cell, the number of gold particles associated with each antigen could be found (labeling efficiency).

Further studies on section thickness measurement.

A rapid, simple, accurate and reproducible technique for measuring section thickness using an instrument known as a surfometer is described. It has shown that microtome settings cannot be relied upon as a means of monitoring the thickness of sections of quenched or wax-embedded tissue. However, there appeared to be a linear relationship between microtome setting and the thickness of dewaxed sections. Microtome settings seem to provide an accurate indication of the thickness of araldite sections.

A method for preparing absolute standards for quantitative calibration and measurement of section thickness with X-ray microanalysis of biological ultrathin specimens in EMMA.

Specimen thickness is determined using X-ray microanalysis in conjunction with atomic absorption spectrophotometry. Specimens of evaporated metal films and standards comprising fixed concentrations of element (such as potassium) in organic media are used to determine elemental concentrations of endogenous elements in embedding resin, from which section thickness can also be determined. The method finally depends only on the single measurement of X-ray emission of such an element in the specimen. Calibration of the X-ray detector over the energy range with mineral salts enables the standard to be used for the quantitative analysis of any element under investigation.

Measurement of section thickness in quantitative microscopy with special reference to enzyme histochemistry.

Quantitative histochemical techniques demand constancy in sectioning tissue and preferably knowledge of the true thickness of a section. In this investigation a simple, rapid, accurate, and reproducible technique is described for measuring section thickness using an instrument known as a Surfometer. It has been shown that: (a) cryostat microtome settings bear little relationship to the true section thickness of the sections produced; (b) that different instruments differ in their inaccuracies; (c) that this inaccuracy varies slightly with different tissues.If densitometric methods are used and either the absolute amount of a tissue component is to be measured or the amount of a tissue component is to be compared in two tissues then it is mandatory to determine the actual thickness of the section. The method described here seems the most appropriate one for these investigations.

Measurement of protein concentration by quantitative electron microscopy.

The method of quantitative electron microscopy was applied to the measurement of protein concentration in thin sections. The human erythrocyte was selected as a model because of its apparently uniform protein concentration. Phosphotungstic acid (PTA) in aqueous solution was used as a reversible stain for protein, and PTA-stained Dowex resin spheres were embedded along with the red cells as standards for measurement of section thickness. The mass of stain removed from a given area of sectioned red cell by buffer (pH 7.4) was measured by quantitative electron microscopy. From the stoichiometry of the reaction between PTA and red cell protein established in this study, the amount of protein present in the measured area was calculated. From this amount of protein and the measured thickness, the concentration of protein was calculated and expressed as g/100 ml, for comparison with the clinical laboratory value for hemoglobin. Groups of red cells from the same sample were measured on 3 different days and their mean values (g/100 ml +/- SD) were 29 +/- 3.9, 30 +/- 2.7, and 33 +/- 4.6, compared to the clinical laboratory value of 32.1 g/100 ml packed cells, after correction for volume change and protein loss during fixation.