Combined Confocal Microscopy and Stereology: a Highly Efficient and Unbiased Approach to Quantitative Structural Measurement in Tissues

Abstract

Understanding the relationship of the structure of organs to their function is a key component of integrative physiological research. The structure of the organs of the body is not constant but changes, both during growth and development and under conditions of sustained stress (e.g. high altitude exposure and disease). Recently, powerful new techniques have become available in molecular biology, which promise to provide novel insights into the mechanisms and consequences of these altered structure‐function relationships. Conventionally structure‐function relationships are studied by microscopic examination of tissue sections. However, drawing conclusions about the three‐dimensional structure of an organ based on this two‐dimensional information frequently leads to serious errors. The techniques of stereology allow precise and accurate quantification of structural features within three‐dimensional organs that relate in a meaningful way to integrated function. For example, knowledge of changes in the total surface area of the capillary endothelium in an organ can be related directly to changes in fluid filtration and permeability, or knowledge of total vessel length and mean radius allows deductions about vascular resistance. Confocal microscopy adds enormously to the power of stereological approaches. It reduces the difficulties and labour involved in obtaining suitable images. Moreover, when used in conjunction with new analytical software, it allows convenient application of stereology to small samples and those in which it is essential to maintain a specific orientation for interpretation. The information obtained will allow us to examine in a quantitative manner the altered structure‐function relationships produced by manipulation of single genes and regulatory pathways in whole organisms.

Local tissue reactions after nerve repair with ethyl-cyanoacrylate compared with epineural sutures

“…Drawing conclusions about a three-dimensional structure of an organ based on two-dimensional information is associated with insecurity and leads to inaccuracy. Confocal microscopy allows a convenient application of stereological techniques to obtain reliable and unbiased measurement of three-dimensional variables from a small sample of nerve tissue [22]. The quantitative analysis of axonal and macrophage profiles used in the present study is based on a modification of the point-counting technique [20], an unbiased stereological technique that is independent of variations in shape and intensity of staining as long as all profiles are identifiable by the investigator, and it is far less time-consuming than total counting.…”

Quiescent adult neural stem cells are exceptionally sensitive to cosmic radiation

“…Quantitative analysis of cell populations was performed by means of design-based (assumption free, unbiased) stereology (Howell et al, 2002;Peterson, 1999). Slices were collected using systematic-random sampling.…”

Quantification of sweat gland innervation

“…Thus the estimated length of nerve fibers within a given area is expressed by the following equation: L E ϭ (T A /T L ϫ ⌺I)/(M ϫ SG % ). 16,17 In this study the T A /T L ϭ 3.05 cm, M ϭ 661ϫ, magnification and SG % ϭ 40% (the biopsies had 50% percent of the tissue sections imaged, 100% of the area sampled, and 80% of the depth of field sampled). The manual quantification of sweat gland nerve fiber density by light microscopy involves taking a digital image of the sweat gland at 20ϫ magnification, shown in G. The same image, taken out of focus, is shown in H, with the selected area of interest highlighted in green.…”

“…As a gold standard, Z-stack confocal images of all the sweat glands in three skin biopsies were analyzed by an unbiased stereologic technique using systematic sampling with a cycloid test system to estimate sweat gland nerve fiber length ( figure 2, A-F). [15][16][17][18][19] Nerve fiber quantitation: Light microscopy. Biopsies underwent blinded IENFD counting and results were expressed as fibers per millimeter.…”