Larsen JO, Gundersen HJ, Nielsen J
Stereological Research Laboratory, University of Aarhus, Denmark; Institute of Pathology, Aalborg Hospital, Aalborg, Denmark; Center for Sensory-Motor Interaction, Aalborg University, Aalborg, Denmark.
J Microsc. 1998 Sep;191(3):238-248. doi: 10.1046/j.1365-2818.1998.00365.x.
Existing design-based direct length estimators require random rotation around at least one axis of the tissue specimen prior to sectioning to ensure isotropy of test probes. In some tissue it is, however, difficult or even impossible to define the region of interest, unless the tissue is sectioned in a specific, nonrandom orientation. Spatial uniform sampling with isotropic virtual planes circumvents the use of physically isotropic or vertical sections. The structure that is contained in a thick physical section is investigated with software-randomized isotropic virtual planes in volume probes in systematically sampled microscope fields using computer-assisted stereological analysis. A fixed volume of 3D space in each uniformly sampled field is probed with systematic random, isotropic virtual planes by a line that moves across the computer screen showing live video images of the microscope field when the test volume is scanned with a focal plane. The intersections between the linear structure and the virtual probes are counted with columns of two dimensional disectors. Global spatial sampling with sets of isotropic uniform random virtual planes provides a basis for length density estimates from a set of parallel physical sections of any orientation preferred by the investigator, i.e. the simplest sampling scheme in stereology. Additional virtues include optimal conditions for reducing the estimator variance, the possibility to estimate total length directly using a fractionator design and the potential to estimate efficiently the distribution of directions from a set of parallel physical sections with arbitrary orientation. Other implementations of the basic idea, systematic uniform sampling using probes that have total 3D x 4pi freedom inside the section, and therefore independent of the position and the orientation of the physical section, are briefly discussed.
现有的基于设计的直接长度估计器要求在切片之前围绕组织标本的至少一个轴进行随机旋转，以确保测试探针的各向同性。然而，在某些组织中，除非以特定的、非随机的方向进行切片，否则很难甚至不可能定义感兴趣的区域。使用各向同性虚拟平面的空间均匀采样避免了使用物理上各向同性或垂直的切片。通过计算机辅助立体分析，在系统采样的显微镜视野中的体积探针中，用软件随机化的各向同性虚拟平面研究厚物理切片中包含的结构。当用焦平面扫描测试体积时，通过在计算机屏幕上移动的一条线，用系统随机的、各向同性的虚拟平面探测每个均匀采样视野中的固定体积的三维空间，该线显示显微镜视野的实时视频图像。用二维分离体列计数线性结构与虚拟探针之间的交点。用各向同性均匀随机虚拟平面集进行全局空间采样，为从研究者选择的任何方向的一组平行物理切片估计长度密度提供了基础，即立体学中最简单的采样方案。其他优点包括降低估计器方差的最佳条件、使用分样器设计直接估计总长度的可能性以及从一组任意方向的平行物理切片有效估计方向分布的潜力。还简要讨论了基本思想的其他实现方式，即使用在切片内部具有完全三维×4π自由度的探针进行系统均匀采样，因此与物理切片的位置和方向无关。
