Systematic random uniform sampling is recommended as the easiest and most efficient way to ensure that the selection of sampling sites is not biased. The first aspect is that every step that reduces the sample size ( from the selection of tissue blocks, tissue sections and microscope fields of view, to where events will be counted) must be carried out in such a way that each portion has an equal chance to be sampled. With regards to sampling, the authors stress two aspects. Thus, stereology provides biologically meaningful 3D data. Practical stereology is the application of unbiased sampling and measurement principles in order to obtain quantitative data about structures in 3D based on nearly 2D (physical or virtual) sections through the structures by using 3D (or geometric) probes. Ochs and Mühlfeld define stereology as “the science of sampling structures with geometric probes.” The purpose of their review is to “provide specific recommendations for the application of stereology to particular animal models of lung disease such as acute lung injury, lung fibrosis, emphysema, pulmonary hypertension, and asthma” (Ochs & Mühlfeld,“Stereology is the gold standard for lung morphometry”). The solutions to both sampling properly and interpreting lower dimensional data are provided by unbiased stereology.
(Ochs & Mühlfeld, “Problems of quantitative microscopy”) a volume is represented by an area (the larger a structure the larger the area it occupies on a section), the surface area is represented by a line (the larger the surface area of a structure the longer its boundary line on a section), the length is represented by the number of transects (the longer a structure the higher is its chance to be seen as a transect in a section) and the number of a structure is simply not represented within one two-dimensional section. Second, each structure we observe under the microscope has ‘lost a dimension.=’: Hence they have to be distributed randomly over the whole organ to make sure that each part has an equal chance of being selected and analyzed. For instance, there is a tendency to sample where the lesions are located during the study of disease: the problem of size reduction warrants that the chosen samples need to represent the whole organ. First, only a small fraction of any organ can be sampled under the microscope, and this can lead to bias. Two problems that can occur during quantitative microscopy are highlighted: sampling and the loss of a dimension. In the next step, it should be defined how these changes can be expressed by simple quantitative parameters, such as cell number for hyperplasia, mean volume for cellular hypertophy, surface area of gas-exchange area, length and number of blood vessels for angiogenesis.” (Ochs & Mühlfeld, “Dissecting lung structure by stereology”).
hyperplasia of airway smooth muscle cells, hypertrophy of alveolar epithelial type II cells, loss of gas-exhange area, angiogenesis of peribronchial blood vessels. The data to be collected must be well understood and defined: “target parameters should be defined as endpoints, e.g.
(Ochs & Mühlfeld, “The challenges of measuring lung structure by microscopy, and how to handle them by stereology” para. Regarding the lung, such parameters may be the volume of alveolar septal tissue, the surface of the alveolar epithelium, the length of nerve fibers innervating the conducting airways or the number of alveoli. The basic parameters that describe the internal lung structure in quantitative terms are characterized by their dimensions: volume (dimension 3), surface (dimension 2), length (dimension 1), number (dimension 0). Physiology, 305(1):L15-22.This paper by Ochs Mühlfeld ( 2013) is an excellent guide for using unbiased stereology to estimate the first order characteristics of the lung: Mühlfeld (2013) Quantitative Microscopy of the Lung – A Problem-Based Approach Part 1: Basic Principles of Lung Stereology.