The Salk Institute for Biological Studies, La Jolla, California 92037, USA.
J Comp Neurol. 2011 Oct 1;519(14):2803-10. doi: 10.1002/cne.22652.
Increasing numbers of neuroanatomists are using stereological methods, and unbiased stereological estimation rules recommend the use of guard zones with the optical disector method to count objects of interest within a volume. Although these methods are statistically unbiased, we believe there is a need to explore sources of systematic bias (e.g., effects of tissue processing and sectioning) that may be affecting estimates of object number. Toward this end, we evaluated neuron distribution through, and tissue shrinkage in, non-embedded tissue cut on a freezing microtome. Our data show that in the x- and y-planes there are minimal changes in tissue area during tissue processing, sectioning, and staining. In the z-axis (perpendicular to the cutting surface), however, sections shrink to ∼25% of the cut thickness. This z-axis shrinkage was quite variable between sections (coefficient of variation about 10%) but stable within the same section (coefficient of variation about 3%). Lastly, individual particle densities are non-uniform through the thickness of the section when the densities should have been uniform. We advise experimenters to use a new protocol, a modified optical disector, for estimation when objects to be counted are marked such that the x-, y-, and z-coordinates are recorded through the full thickness of a section and guard zones are applied post data collection based on the characteristics of the object distribution along the z-axis. It is likely that individual experiments with different embedding materials and histological processing steps could require guard zones of varying sizes, or none at all, depending on object distribution in the z-axis.
越来越多的神经解剖学家正在使用体视学方法，无偏体视学估计规则建议使用光学切割器方法中的保护带，以对体积内的感兴趣物体进行计数。虽然这些方法在统计学上是无偏的，但我们认为有必要探索可能影响物体数量估计的系统偏差来源（例如，组织处理和切片的影响）。为此，我们评估了通过冷冻切片机切割的非包埋组织中的神经元分布和组织收缩。我们的数据表明，在 x 和 y 平面上，组织处理、切片和染色过程中组织面积的变化最小。然而，在 z 轴（垂直于切割面）上，切片收缩到约切割厚度的 25%。这种 z 轴收缩在切片之间变化很大（变异系数约为 10%），但在同一切片内稳定（变异系数约为 3%）。最后，当颗粒密度应该均匀时，个体颗粒密度在切片的厚度上不均匀。我们建议实验者在对标记的要计数的物体进行估计时使用新的方案，即改良的光学切割器，当 x、y 和 z 坐标通过切片的整个厚度记录，并且根据物体沿 z 轴分布的特征在数据收集后应用保护带。可能需要根据不同的包埋材料和组织处理步骤，为 z 轴上的物体分布分配不同大小的保护带，或者根本不需要保护带。
